Compositions comprising circular polyribonucleotides and uses thereof

ABSTRACT

This invention relates generally to pharmaceutical compositions and preparations of circular polyribonucleotides and uses thereof.

CROSS-REFERENCE

This application is a continuation of U.S. patent application Ser. No.17/173,991, filed Feb. 11, 2021, which is a continuation of U.S. patentapplication Ser. No. 16/438,073, filed on Jun. 11, 2019, now U.S. Pat.No. 10,953,033, issued Mar. 23, 2021, which is a continuation ofInternational Application No. PCT/US2018/065836, filed on Dec. 14, 2018,which claims the benefit of U.S. Provisional Application No. 62/599,547,filed on Dec. 15, 2017, and U.S. Provisional Application No. 62/676,688,filed on May 25, 2018, each of which are incorporated herein byreference in their entirety.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted electronically in ASCII format and is hereby incorporated byreference in its entirety. Said ASCII copy, created on Jan. 28, 2019, isnamed 29927-801_601_SL.txt and is 71,828 bytes in size.

BACKGROUND

Certain circular polyribonucleotides are ubiquitously present in humantissues and cells, including tissues and cells of healthy individuals.

SUMMARY

In one aspect, the invention includes a pharmaceutical compositioncomprising a circular polyribonucleotide that comprises at least onestructural element selected from a) an encryptogen; b) a staggerelement; c) a regulatory element; d) a replication element; f)quasi-double-stranded secondary structure; and g) expression sequence;and at least one functional characteristic selected from: a) greatertranslation efficiency than a linear counterpart; b) a stoichiometrictranslation efficiency of multiple translation products; c) lessimmunogenicity than a counterpart lacking an encryptogen; d) increasedhalf-life over a linear counterpart; and e) persistence during celldivision.

In some embodiments, the circular polyribonucleotide is translationcompetent. In one such embodiment, the quasi-helical structure comprisesat least one double-stranded RNA segment with at least onenon-double-stranded segment. In another such embodiment, thequasi-helical structure comprises a first sequence and a second sequencelinked with a repetitive sequence, e.g., an A-rich sequence.

In some embodiments, the circular polyribonucleotide comprises anencryptogen. In some embodiments, the encryptogen comprises at least onemodified ribonucleotide, e.g., pseudo-uridine, N(6)methyladenosine(m6A). In some embodiments, the encryptogen comprises a protein bindingsite, e.g., ribonucleotide binding protein. In some embodiments, theencryptogen comprises an immunoprotein binding site, e.g., to evadeimmune reponses, e.g., CTL responsess.

In some embodiments, the circular polyribonucleotide comprises at leastone modified ribonucleotide.

In some embodiments, the circular polyribonucleotide has at least 2×less immunogenicity than a counterpart lacking the encryptogen, e.g., asassessed by expression or signaling or activation of at least one ofRIG-I, TLR-3, TLR-7, TLR-8, MDA-5, LGP-2, OAS, OASL, PKR, IFN-beta.

In some embodiments, the circular polyribonucleotide further comprises ariboswitch.

In some embodiments, the circular polyribonucleotide further comprisesan aptazyme.

In some embodiments, the circular polyribonucleotide comprises atranslation initiation sequence, e.g., GUG, CUG start codon, e.g.,expression under stress conditions.

In some embodiments, the circular polyribonucleotide comprises at leastone expression sequence, e.g., encoding a polypeptide. In one suchembodiments, the expression sequence encodes a peptide orpolynucleotide. In some embodiments, the circular polyribonucleotidecomprises a plurality of expression sequences, either the same ordifferent.

In some embodiments, the circular polyribonucleotide comprises a staggerelement, e.g., 2A.

In some embodiments, the circular polyribonucleotide comprises aregulatory nucleic acid, e.g., a non-coding RNA. In some embodiments,the circular polyribonucleotide comprises a regulatory element, e.g.,that alters expression of an expression sequence.

In some embodiments, the circular polyribonucleotide has a size in therange of about 20 bases to about 20 kb.

In some embodiments, the circular polyribonucleotide is synthesizedthrough circularization of a linear polynucleotide.

In some embodiments, the circular polyribonucleotide is substantiallyresistant to degradation, e.g., exonuclease.

In some embodiments, the circular polyribonucleotide lacks at least oneof: a) a 5′-UTR; b) a 3′-UTR; c) a poly-A sequence; d) a 5′-cap; e) atermination element; f) an internal ribosomal entry site; g) degradationsusceptibility by exonucleases and h) binding to a cap-binding protein.

In one aspect, the invention includes a method of producing thecomposition comprising a circular polyribonucleotide described herein.

In one aspect, the invention includes a pharmaceutical compositioncomprising a pharmaceutically acceptable carrier or excipient and acircular polyribonucleotide that comprises one or more expressionsequences, wherein the circular polyribonucleotide is competent forrolling circle translation.

In some embodiments, each of the one or more expression sequences isseparated from a succeeding expression sequence by a stagger element inthe circular polyribonucleotide, wherein rolling circle translation ofthe one or more expression sequences generates at least two polypeptidemolecules, e.g., the stagger elements stalls or halts the ribosome suchthat the elongating polypeptide falls off the ribosome. In someembodiments, the stagger element prevents generation of a singlepolypeptide (a) from two rounds of translation of a single expressionsequence or (b) from one or more rounds of translation of two or moreexpression sequences. For example, the stagger element can preventgeneration of a single polypeptide from two or more rounds oftranslation of two or more expression sequences, e.g., the staggerelement halts the ribosome and/or allows the elongating polypeptide tofall off the ribosome after one circuit around the circularpolyribonucleotide.

In some embodiments, the stagger element is a sequence separate from theone or more expression sequences.

In some embodiments, the stagger element comprises a portion of anexpression sequence of the one or more expression sequences.

In one aspect, the invention includes a pharmaceutical compositioncomprising a pharmaceutically acceptable carrier or excipient and acircular polyribonucleotide that comprises one or more expressionsequences and is competent for rolling circle translation, wherein thecircular polyribonucleotide is configured such that at least 10%, 20%,30%, 40%, 50%, at least 60%, at least 70%, at least 80%, at least 90%,at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or100% of total polypeptides (molar/molar) generated during the rollingcircle translation of the circular polyribonucleotide are discretepolypeptides, and wherein each of the discrete polypeptides is generatedfrom a single round of translation or less than a single round oftranslation of the one or more expression sequences.

In some embodiments, the circular polyribonucleotide is configured suchthat at least 10%, 20%, 30%, 40%, 50%, at least 60%, at least 70%, atleast 80%, at least 90%, at least 95%, at least 96%, at least 97%, atleast 98%, at least 99%, or 100% of total polypeptides (molar/molar)generated during the rolling circle translation of the circularpolyribonucleotide are the discrete polypeptides, and wherein amountratio of the discrete products over the total polypeptides is tested inan in vitro translation system.

In some embodiments, the in vitro translation system comprises rabbitreticulocyte lysate.

In some embodiments, the stagger element is downstream of or 3′ to atleast one of the one or more expression sequences, wherein the staggerelement is configured to stall a ribosome during rolling circletranslation of the circular polyribonucleotide.

In one aspect, the invention provides a pharmaceutical compositioncomprising a pharmaceutically acceptable carrier or excipient and acircular polyribonucleotide that comprises one or more expressionsequences and a stagger element downstream of or 3′ to at least one ofthe one or more expression sequences. In some embodiments, the staggerelement is configured to stall a ribosome during rolling circletranslation of the circular polyribonucleotide.

In some embodiments, the stagger element encodes a peptide sequenceselected from the group consisting of a 2A sequence and a 2A-likesequence.

In some embodiments, the stagger element encodes a sequence with aC-terminal sequence that is GP.

In some embodiments, the stagger element encodes a sequence with aC-terminal consensus sequence that is D(V/I)ExNPG P (SEQ ID NO: 61),where x=any amino acid.

In some embodiments, the stagger element encodes at least one of ofGDVESNPGP (SEQ ID NO: 62), GDIEENPGP (SEQ ID NO: 63), VEPNPGP (SEQ IDNO: 64), IETNPGP (SEQ ID NO: 65), GDIESNPGP (SEQ ID NO: 66), GDVELNPGP(SEQ ID NO: 67), GDIETNPGP (SEQ ID NO: 68), GDVENPGP (SEQ ID NO: 69),GDVEENPGP (SEQ ID NO: 70), GDVEQNPGP (SEQ ID NO: 71), IESNPGP (SEQ IDNO: 72), GDIELNPGP (SEQ ID NO: 73), HDIETNPGP (SEQ ID NO: 74), HDVETNPGP(SEQ ID NO: 75), HDVEMNPGP (SEQ ID NO: 76), GDMESNPGP (SEQ ID NO: 77),GDVETNPGP (SEQ ID NO: 78), GDIEQNPGP (SEQ ID NO: 79), and DSEFNPGP (SEQID NO: 80).

In some embodiments, the stagger element is downstream of or 3′ to eachof the one or more expression sequences.

In some embodiments, the stagger element of a first expression sequencein the circular polyribonucleotide is upstream of (5′ to) a firsttranslation initiation sequence of an expression sequence succeeding thefirst expression sequence in the circular polyribonucleotide, andwherein a distance between the stagger element and the first translationinitiation sequence enables continuous translation of the firstexpression sequence and the succeeding expression sequence. In someembodiments, the stagger element comprises a termination element of afirst expression sequence on the circular polyribonucleotide that has adistance upstream from (5′ to) a translation initiation sequence of anexpression sequence succeeding the first expression sequence on thecircular polyribonucleotide, and wherein the distance enables continuoustranslation of the first expression sequence and its succeedingexpression sequence.

In some embodiments, a first stagger element is upstream of (5′ to) afirst translation initiation sequence of a first expression sequence inthe circular polyribonucleotide, wherein the circular polyribonucleotideis continuously translated, wherein a corresponding circularpolyribonucleotide comprises a second stagger element upstream of asecond translation initiation sequence of a second expression sequencein the corresponding circular polyribonucleotide that is notcontinuously translated, and wherein the second stagger element in thecorresponding circular polyribonucleotide is at a greater distance fromthe second translation initiation sequence, e.g., at least 2×, 3×, 4×,5×, 6×, 7×, 8×, 9×, 10×, than a distance between the first staggerelement and the first translation initiation in the circularpolyribonucleotide. In some embodiments, the stagger element comprises afirst termination element upstream of (5′ to) a first translationinitiation sequence of a first expression sequence in the circularpolyribonucleotide, wherein the circular polyribonucleotide iscontinuously translated and a corresponding circular polyribonucleotidecomprises a stagger element comprising a second termination elementupstream from a second translation initiation sequence of a secondexpression sequence in the corresponding circular polyribonucleotidethat is not continuously translated, and where the second terminationelement in the corresponding circular polyribonucleotide is at a greaterdistance from the second translation initiation sequence, e.g., at least2×, 3×, 4×, 5×, 6×, 7×, 8×, 9×, 10×, than a distance between the firsttermination element and the first translation initiation in the circularpolyribonucleotide.

In some embodiments, the distance between the first stagger element andthe first translation initiation is at least 2 nt, 3 nt, 4 nt, 5 nt, 6nt, 7 nt, 8 nt, 9 nt, 10 nt, 11 nt, 12 nt, 13 nt, 14 nt, 15 nt, 16 nt,17 nt, 18 nt, 19 nt, 20 nt, 25 nt, 30 nt, 35 nt, 40 nt, 45 nt, 50 nt, 55nt, 60 nt, 65 nt, 70 nt, 75 nt, or greater. In some embodiments, thedistance between the second stagger element and the second translationinitiation is at least 2 nt, 3 nt, 4 nt, 5 nt, 6 nt, 7 nt, 8 nt, 9 nt,10 nt, 11 nt, 12 nt, 13 nt, 14 nt, 15 nt, 16 nt, 17 nt, 18 nt, 19 nt, 20nt, 25 nt, 30 nt, 35 nt, 40 nt, 45 nt, 50 nt, 55 nt, 60 nt, 65 nt, 70nt, 75 nt, or greater than the distance between the first staggerelement and the first translation initiation.

In some embodiments, the circular polyribonucleotide comprises more thanone expression sequence.

In some embodiments, the circular polyribonucleotide has a translationefficiency at least 5%, at least 10%, at least 15%, at least 20%, atleast 30%, at least 40%, at least 50%, at least 60%, at least 70%, atleast 80%, at least 90%, at least 100%, at least 150%, at least 2 fold,at least 3 fold, at least 4 fold, at least 5 fold, at least 6 fold, atleast 7 fold, at least 8 fold, at least 9 fold, at least 10 fold, atleast 20 fold, at least 50 fold, or at least 100 fold greater than alinear counterpart.

In some embodiments, the circular polyribonucleotide has a translationefficiency at least 5 fold greater than a linear counterpart.

In some embodiments, the circular polyribonucleotide lacks an internalribosomal entry site.

In some embodiments, the one or more expression sequences comprise aKozak initiation sequence.

In some embodiments, the one or more expression sequences encodes apeptide.

In some embodiments, the circular polyribonucleotide comprises aregulatory nucleic acid, e.g., a non-coding RNA. In some embodiments,the circular polyribonucleotide comprises a regulatory element, e.g.,that alters expression of an expression sequence.

In one aspect, the invention provides a circular polyribonucleotide ofany of the pharmaceutical composition provided herein.

In one aspect, the invention includes a method of producing thepharmaceutical composition provided herien, comprising combining thecircular polyribonucleotide described herein and the pharmaceuticallyacceptable carrier or excipient described herein.

In one aspect, the invention includes a method of administering thecomposition comprising a circular polyribonucleotide described herein.

In one aspect, the invention includes a method for protein expression,comprising translating at least a region of the circularpolyribonucleotide provided herein.

In some embodiments, the translation of at least a region of thecircular polyribonucleotide takes place in vitro. In some embodiments,the translation of the at least a region of the circularpolyribonucleotide takes place in vivo.

In one aspect, the invention includes a polynucleotide, e.g., a DNAvector, encoding the circular polyribonucleotide provided herein.

In one aspect, the invention includes a method of producing the circularpolyribonucleotide as provided herein.

In some embodiments, the method comprises splint ligation-mediatedcircularization of a linear polyribonucleotide.

In some embodiments, the circularization, e.g., splint ligation-mediatedcircularization, has an efficiency of at least 2%, at least 5%, at least10%, at least 15%, at least 20%, at least 25%, at least 30%, at least32%, at least 34%, at least 36%, at least 38%, at least 40%, at least42%, at least 44%, at least 46%, at least 48%, or at least 50%. In someembodiments, the splint ligation-mediated circularization has anefficiency of about 40% to about 50% or more than 50%.

Definitions

The present invention will be described with respect to particularembodiments and with reference to certain figures but the invention isnot limited thereto but only by the claims. Terms as set forthhereinafter are generally to be understood in their common sense unlessindicated otherwise.

The terms “obtainable by”, “producable by” or the like are used toindicate that a claim or embodiment refers to compound, composition,product, etc. per se, i.e. that the compound, composition, product, etc.can be obtained or produced by a method which is described formanufacture of the compound, composition, product, etc., but that thecompound, composition, product, etc. may be obtained or produced byother methods than the described one as well. The terms “obtained by”,“produced by” or the like indicate that the compound, composition,product, is obtained or produced by a recited specific method. It is tobe understood that the terms “obtainable by”, “producable by” and thelike also disclose the terms “obtained by”, “produced by” and the likeas a preferred embodiment of “obtainable by”, “producible by” and thelike.

The wording “compound, composition, product, etc. for treating,modulating, etc.” is to be understood to refer a compound, composition,product, etc. per se which is suitable for the indicated purposes oftreating, modulating, etc. The wording “compound, composition, product,etc. for treating, modulating, etc.” additionally discloses that, as apreferred embodiment, such compound, composition, product, etc. is foruse in treating, modulating, etc.

The wording “compound, composition, product, etc. for use in . . . ” or“use of a compound, composition, product, etc in the manufacture of amedicament, pharmaceutical composition, veterinary composition,diagnostic composition, etc. for . . . ” indicates that such compounds,compositions, products, etc. are to be used in therapeutic methods whichmay be practiced on the human or animal body. They are considered as anequivalent disclosure of embodiments and claims pertaining to methods oftreatment, etc. If an embodiment or a claim thus refers to “a compoundfor use in treating a human or animal being suspected to suffer from adisease”, this is considered to be also a disclosure of a “use of acompound in the manufacture of a medicament for treating a human oranimal being suspected to suffer from a disease” or a “method oftreatment by administering a compound to a human or animal beingsuspected to suffer from a disease”. The wording “compound, composition,product, etc. for treating, modulating, etc.” is to be understood torefer a compound, composition, product, etc. per se which is suitablefor the indicated purposes of treating, modulating, etc.

The term “pharmaceutical composition” is intended to also disclose thatthe circular polyribonucleotide comprised within a pharmaceuticalcomposition can be used for the treatment of the human or animal body bytherapy. It is thus meant to be equivalent to the “a circularpolyribonucleotide for use in therapy”.

The circular polyribonucleotides, compositions comprising such circularpolyribonucleotides, methods using such circular polyribonucleotides,etc. as described herein are based in part on the examples whichillustrate how circular polyribonucleotides effectors comprisingdifferent elements, for example a replication element, an expressionsequence, a stagger element and an encryptogen (see e.g., example 1) orfor example an expression sequences, a stagger element and a regulatoryelement (see e.g., examples 30 and 38) can be used to achieve differenttechnical effects (e.g. increased translation efficiency than a linearcounterpart in examples 1 and 38 and increased half-life over a linearcounterpart in example 38). It is on the basis of inter alia theseexamples that the description hereinafter contemplates variousvariations of the specific findings and combinations considered in theexamples.

As used herein, the terms “circRNA” or “circular polyribonucleotide” or“circular RNA” are used interchangeably and can refer to apolyribonucleotide that forms a circular structure through covalent ornon-covalent bonds.

As used herein, the term “encryptogen” can refer to a nucleic acidsequence or structure of the circular polyribonucleotide that aids inreducing, evading, and/or avoiding detection by an immune cell and/orreduces induction of an immune response against the circularpolyribonucleotide.

As used herein, the term “expression sequence” can refer to a nucleicacid sequence that encodes a product, e.g., a peptide or polypeptide, ora regulatory nucleic acid. An exemplary expression sequence that codesfor a peptide or polypeptide can comprise a plurality of nucleotidetriads, each of which can code for an amino acid and is termed as a“codon”.

As used herein, the term “immunoprotein binding site” can refer to anucleotide sequence that binds to an immunoprotein. In some embodiments,the immunoprotein binding site aids in masking the circularpolyribonucleotide as exogenous, for example, the immunoprotein bindingsite can be bound by a protein (e.g., a competitive inhibitor) thatprevents the circular polyribonucleotide from being recognized and boundby an immunoprotein, thereby reducing or avoiding an immune responseagainst the circular polyribonucleotide. As used herein, the term“immunoprotein” can refer to any protein or peptide that is associatedwith an immune response, e.g., such as against an immunogen, e.g., thecircular polyribonucleotide. Non-limiting examples of immunoproteininclude T cell receptors (TCRs), antibodies (immunoglobulins), majorhistocompatibility complex (MHC) proteins, complement proteins, and RNAbinding proteins.

As used herein, the term “modified ribonucleotide” can refer to anucleotide with at least one modification to the sugar, the nucleobase,or the internucleoside linkage.

As used herein, the phrase “quasi-helical structure” can refer to ahigher order structure of the circular polyribonucleotide, wherein atleast a portion of the circular polyribonucleotide folds into a helicalstructure.

As used herein, the phrase “quasi-double-stranded secondary structure”can refer to a higher order structure of the circularpolyribonucleotide, wherein at least a portion of the circularpolyribonucleotide creates an internal double strand.

As used herein, the term “regulatory element” can refer to a moiety,such as a nucleic acid sequence, that modifies expression of anexpression sequence within the circular polyribonucleotide.

As used herein, the term “repetitive nucleotide sequence” can refer to arepetitive nucleic acid sequence within a stretch of DNA or RNA orthroughout a genome. In some embodiments, the repetitive nucleotidesequence includes poly CA or poly TG (UG) sequences. In someembodiments, the repetitive nucleotide sequence includes repeatedsequences in the Alu family of introns.

As used herein, the term “replication element” can refer to a sequenceand/or motifs useful for replication or that initiate transcription ofthe circular polyribonucleotide.

As used herein, the term “stagger element” can refer to a moiety, suchas a nucleotide sequence, that induces ribosomal pausing duringtranslation. In some embodiments, the stagger element is a non-conservedsequence of amino-acids with a strong alpha-helical propensity followedby the consensus sequence −D(V/I)ExNPG P (SEQ ID NO: 61), where x=anyamino acid. In some embodiments, the stagger element may include achemical moiety, such as glycerol, a non nucleic acid linking moiety, achemical modification, a modified nucleic acid, or any combinationthereof.

As used herein, the term “substantially resistant” can refer to one thathas at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%,98% or 99% resistance as compared to a reference.

As used herein, the term “stoichiometric translation” can refer to asubstantially equivalent production of expression products translatedfrom the circular polyribonucleotide. For example, for a circularpolyribonucleotide having two expression sequences, stoichiometrictranslation of the circular polyribonucleotide can mean that theexpression products of the two expression sequences can havesubstantially equivalent amounts, e.g., amount difference between thetwo expression sequences (e.g., molar difference) can be about 0, orless than 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, or 20%.

As used herein, the term “translation initiation sequence” can refer toa nucleic acid sequence that initiates translation of an expressionsequence in the circular polyribonucleotide.

As used herein, the term “termination element” can refer to a moiety,such as a nucleic acid sequence, that terminates translation of theexpression sequence in the circular polyribonucleotide.

As used herein, the term “translation efficiency” can refer to a rate oramount of protein or peptide production from a ribonucleotidetranscript. In some embodiments, translation efficiency can be expressedas amount of protein or peptide produced per given amount of transcriptthat codes for the protein or peptide, e.g., in a given period of time,e.g., in a given translation system, e.g., an in vitro translationsystem like rabbit reticulocyte lysate, or an in vivo translation systemlike a eukaryotic cell or a prokaryotic cell.

As used herein, the term “circularization efficiency” can refer to ameasurement of resultant circular polyribonucleotide versus its startingmaterial.

As used herein, the term “immunogenic” can refer to a potential toinduce an immune response to a substance. In some embodiments, an immuneresponse may be induced when an immune system of an organism or acertain type of immune cells is exposed to an immunogenic substance. Theterm “non-immunogenic” can refer to a lack of or absence of an immuneresponse above a detectable threshold to a substance. In someembodiments, no immune response is detected when an immune system of anorganism or a certain type of immune cells is exposed to anon-immunogenic substance. In some embodiments, a non-immunogeniccircular polyribonucleotide as provided herein, does not induce animmune response above a pre-determined threshold when measured by animmunogenicity assay. For example, when an immunogenicity assay is usedto measure antibodies raised against a circular polyribonucleotide orinflammatory markers, a non-immunogenic polyribonucleotide as providedherein can lead to production of antibodies or markers at a level lowerthan a predetermined threshold. The predetermined threshold can be, forinstance, at most 1.5 times, 2 times, 3 times, 4 times, or 5 times thelevel of antibodies or markers raised by a control reference.

INCORPORATION BY REFERENCE

All publications, patents, and patent applications mentioned in thisspecification are herein incorporated by reference to the same extent asif each individual publication, patent, or patent application wasspecifically and individually indicated to be incorporated by reference.

BRIEF DESCRIPTION OF THE DRAWINGS

The following detailed description of the embodiments of the inventionwill be better understood when read in conjunction with the appendeddrawings. For the purpose of illustrating the invention, there are shownin the drawings embodiments, which are presently exemplified. It shouldbe understood, however, that the invention is not limited to the precisearrangement and instrumentalities of the embodiments shown in thedrawings.

FIG. 1 shows different exemplary circularization methods.

FIG. 2 shows a schematic of an exemplary in vitro production process ofa circular RNA that contains a start-codon, an ORF (open reading frame)coding for GFP, a stagger element (2A), an encryptogen, and an IRES(internal ribosome entry site).

FIG. 3 shows a schematic of an exemplary in vivo production process of acircular RNA.

FIG. 4 shows design of an exemplary circular RNA that comprises astart-codon, an ORF coding for GFP, a stagger element (2A), and anencryptogen.

FIGS. 5A and 5B are schematics demonstrating in vivo stoichiometricprotein expression of two different circular RNAs.

FIG. 6 shows a schematic of a control circular RNA that has an intronand expresses GFP.

FIG. 7 shows a schematic of an exemplary circular RNA that has asynthetic riboswitch (in red) regulating the expression of the GFP fromthe circular RNA in the presence or absence of ligands to theriboswitch.

FIG. 8 is a schematic demonstrating in vivo protein expression in mousemodel from exemplary circular RNAs.

FIG. 9 is a schematic demonstrating in vivo biodistribution of anexemplary circular RNA in a mouse model.

FIG. 10 is a schematic demonstrating in vivo protein expression in mousemodel from an exemplary circular RNA that harbors an encryptogen(intron).

FIG. 11 shows a schematic of an exemplary circular RNA that has onedouble-stranded RNA segment, which can be subject to dot blot analysisfor its structural information.

FIG. 12 shows a schematic of an exemplary circular RNA that has aqusi-helical structure (HDVmin), which can be subject to SHAPE analysisfor its structural information.

FIG. 13 shows a schematic of an exemplary circular RNA that has afunctional qusi-helical structure (HDVmin), which demonstrates HDAgbinding activity.

FIG. 14 is a schematic demonstrating transcription, self-cleavage, andligation of an exemplary self-replicable circular RNA.

FIG. 15 shows a schematic of an exemplary circular RNA that is expressedin vivo and has improved in vivo stability.

FIG. 16 shows a schematic of an exemplary circular RNA that is preservedduring mitosis and persists in daughter cells. BrdU pulse as shown isused for labeling the divided cells.

FIG. 17 is a denaturing PAGE gel image demonstrating in vitro productionof different exemplary circular RNAs.

FIG. 18 is a graph summarizing circularization efficiencies of differentexemplary circular RNAs.

FIG. 19 is a denaturing PAGE gel image demonstrating decreaseddegradation susceptibility of an exemplary circular RNA as compared toits linear counterpart.

FIG. 20 is a denaturing PAGE gel image demonstrating exemplary circularRNA after an exemplary purification process.

FIG. 21 is a Western blot image demonstrating expression of Flag protein(˜15 kDa) by an exemplary circular RNA that lacks IRES, cap, 5′ and 3′UTRs.

FIG. 22 is Western blot image demonstrating rolling-circle translationof an exemplary circular RNA.

FIG. 23 shows Western blot images demonstrating production of discreteproteins or continuous long peptides from different exemplary circularRNAs with or without an exemplary stagger element.

FIG. 24A is a Western blot image showing the comparison of proteinexpression between different exemplary circular RNAs with an exemplarystagger element or a termination element (stop codon).

FIG. 24B is a graph summarizing the signal intensity from Western blotanalysis of the protein products translated from the two exemplarycircular RNAs.

FIG. 25 is a graph summarizing the luciferase activity of translationproducts of an exemplary circular RNA and its linear counterpart, incomparison with a vehicle control RNA.

FIG. 26 is a graph summarizing RNA quantities at different collectiontime points in a time course experiment testing half-life of anexemplary circular RNA.

FIG. 27A is a graph showing qRT-PCR analysis of linear and circular RNAlevels 24 hours after delivery to cells using primers that captured bothlinear and circular RNA.

FIG. 27B is a graph showing qRT-PCR analysis of linear and circular RNAlevels using a primer specific for the circular RNA.

FIG. 28 is an image showing a blot of cell lysates from circular RNA andlinear RNA probed for EGF protein and a beta-tubulin loading control.

FIG. 29 is a graph showing qRT-PCR analysis of immune related genes from293T cells transfected with circular RNA or linear RNA.

FIG. 30 is a graph showing luciferase activity of protein expressed fromcircular RNA via rolling circle translation.

FIG. 31 is a graph showing luciferase activity of protein expressed fromcircular RNA or linear RNA.

FIG. 32 is a graph showing luciferase activity of protein expressed fromlinear RNA or circular RNA via rolling circle translation.

FIG. 33 is a graph showing luciferase activity of protein expressed fromcircular RNA via IRES translation initiation.

FIG. 34 is a graph showing luciferase activity of protein expressed fromcircular RNA via IRES initiation and rolling circle translation.

FIG. 35 is an image showing a protein blot of expression products fromcircular RNA or linear RNA.

FIG. 36 is an image showing a protein blot of expression products fromcircular RNA or linear RNA.

FIG. 37 shows predicted structure with a quasi-double stranded structureof an exemplary circular RNA.

FIG. 38 shows predicted structure with a quasi-helical structure of anexemplary circular RNA.

FIG. 39 shows predicted structure with a quasi-helical structure linkedwith a repetitive sequence of an exemplary circular RNA.

FIG. 40 demonstrates experimental data that degradation by RNAse H of anexemplary circular RNA produced nucleic acid degradation productsconsistent with a circular and not a concatemeric RNA.

FIG. 41 shows an electrophoresis image of the different lengths of DNAthat were generated for the creation of a wide variety of RNA lengths.

FIG. 42 shows experimental data that confirmed the circularization ofRNAs using RNAse R treatment and qPCR analysis against circularjunctions of a wide variety of lengths.

FIG. 43 shows generation of exemplary circular RNA with a miRNA bindingsite (SEQ ID NOS 112 and 129, respectively, in order of appearance).

FIG. 44 shows generation of exemplary circular RNA by self-splicing.

FIG. 45 shows generation of exemplary circular RNA with a proteinbinding site.

FIG. 46 shows experimental data demonstrating the higher stability ofcircular RNA in a dividing cell as compared to linear controls.

FIG. 47 shows experimental data demonstrating the protein expressionfrom exemplary circular RNAs with a plurality of expression sequencesand the rolling circle translation of exemplary circular RNAs withmultiple expression sequences.

FIG. 48 shows experimental data demonstrating the reduced toxicity totransfected cells of an exemplary circular RNA as compared to linearcontrol.

FIG. 49 shows that exemplary circular RNA was translated at a higherlevel as compared to linear RNA under stress condition.

FIG. 50 shows generation of circular RNAs with a riboswitch.

FIGS. 51A, 51B, and 51C show that the modified circular RNAs weretranslated in cells.

FIGS. 52A-52C show that modified circular RNAs have reducedimmuinogeneicity as compared to unmodified circular RNAs to cells asassessed by MDAS, OAS and IFN-beta expression in the transfected cells.

FIG. 53 shows that after injection into mice, circular RNA was detectedat higher levels than linear RNA in livers of mice at 3, 4, and 7 dayspost-injection.

FIGS. 54A and 54B show that after injection of circular RNA or linearRNA expressing Gaussia Luciferase into mice, Gaussia Luciferase activitywas detected in plasma at 1, 2,7, 11, 16, and 23 days post-dosing ofcircular RNA, while its activity was only detected in plasma at 1, and 2days post-dosing of modified linear RNA.

FIG. 55 show that after injection of RNA, circular RNA but not linearRNA, was detected in the liver and spleen at 16 days post-administrationof RNA.

FIG. 56 show that after injection of RNA, linear RNA but not circularRNA, showed immunogenicity as assessed by RIG-I, MDA-5, IFN-B and OAS.

DETAILED DESCRIPTION

This invention relates generally to pharmaceutical compositions andpreparations of circular polyribonucleotides and uses thereof.

Circular Polyribonucleotides

In some aspects, the invention described herein comprises compositionsand methods of using and making circular polyribonucleotides, anddelivery of circular polyribonucleotides. In some embodiments, thecircular polyribonucleotide is non-immunogenic in a mammal, e.g., ahuman. In some embodiments, the circular polyribonucleotide is capableof replicating or replicates in a cell from an aquaculture animal (fish,crabs, shrimp, oysters etc.), a mammalian cell, e.g., a cell from a petor zoo animal (cats, dogs, lizards, birds, lions, tigers and bearsetc.), a cell from a farm or working animal (horses, cows, pigs,chickens etc.), a human cell, cultured cells, primary cells or celllines, stem cells, progenitor cells, differentiated cells, germ cells,cancer cells (e.g., tumorigenic, metastic), non-tumorigenic cells(normal cells), fetal cells, embryonic cells, adult cells, mitoticcells, non-mitotic cells, or any combination thereof. In someembodiments, the invention includes a cell comprising the circularpolyribonucleotide described herein, wherein the cell is a cell from anaquaculture animal (fish, crabs, shrimp, oysters etc.), a mammaliancell, e.g., a cell from a pet or zoo animal (cats, dogs, lizards, birds,lions, tigers and bears etc.), a cell from a farm or working animal(horses, cows, pigs, chickens etc.), a human cell, a cultured cell, aprimary cell or a cell line, a stem cell, a progenitor cell, adifferentiated cell, a germ cell, a cancer cell (e.g., tumorigenic,metastic), a non-tumorigenic cell (normal cells), a fetal cell, anembryonic cell, an adult cell, a mitotic cell, a non-mitotic cell, orany combination thereof. In some embodiments, the cell is modified tocomprise the circular polyribonucleotide.

In some embodiments, the circular polyribonucleotide includes sequencesor expression products.

In some embodiments, the circular polyribonucleotide has a half-life ofat least that of a linear counterpart, e.g., linear expression sequence,or linear circular polyribonucleotide. In some embodiments, the circularpolyribonucleotide has a half-life that is increased over that of alinear counterpart. In some embodiments, the half-life is increased byabout 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, or greater. Insome embodiments, the circular polyribonucleotide has a half-life orpersistence in a cell for at least about 1 hr to about 30 days, or atleast about 2 hrs, 6 hrs, 12 hrs, 18 hrs, 24 hrs, 2 days, 3, days, 4days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days,13 days, 14 days, 15 days, 16 days, 17 days, 18 days, 19 days, 20 days,21 days, 22 days, 23 days, 24 days, 25 days, 26 days, 27 days, 28 days,29 days, 30 days, 60 days, or longer or any time therebetween. Incertain embodiments, the circular polyribonucleotide has a half-life orpersistence in a cell for no more than about 10 mins to about 7 days, orno more than about 1 hr, 2 hrs, 3 hrs, 4 hrs, 5 hrs, 6 hrs, 7 hrs, 8hrs, 9 hrs, 10 hrs, 11 hrs, 12 hrs, 13 hrs, 14 hrs, 15 hrs, 16 hrs, 17hrs, 18 hrs, 19 hrs, 20 hrs, 21 hrs, 22 hrs, 24 hrs, 36 hrs, 48 hrs, 60hrs, 72 hrs, 4 days, 5 days, 6 days, 7 days, or any time therebetween.In some embodiments, the circular polyribonucleotide has a half-life orpersistence in a cell while the cell is dividing. In some embodiments,the circular polyribonucleotide has a half-life or persistence in a cellpost division. In certain embodiments, the circular polyribonucleotidehas a half-life or persistence in a dividing cell for greater than aboutabout 10 minutes to about 30 days, or at least about 1 hr, 2 hrs, 3 hrs,4 hrs, 5 hrs, 6 hrs, 7 hrs, 8 hrs, 9 hrs, 10 hrs, 11 hrs, 12 hrs, 13hrs, 14 hrs, 15 hrs, 16 hrs, 17 hrs, 18 hrs, 24 hrs, 2 days, 3, days, 4days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days,13 days, 14 days, 15 days, 16 days, 17 days, 18 days, 19 days, 20 days,21 days, 22 days, 23 days, 24 days, 25 days, 26 days, 27 days, 28 days,29 days, 30 days, 60 days, or longer or any time therebetween.

In some embodiments, the circular polyribonucleotide modulates acellular function, e.g., transiently or long term. In certainembodiments, the cellular function is stably altered, such as amodulation that persists for at least about 1 hr to about 30 days, or atleast about 2 hrs, 6 hrs, 12 hrs, 18 hrs, 24 hrs, 2 days, 3, days, 4days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days,13 days, 14 days, 15 days, 16 days, 17 days, 18 days, 19 days, 20 days,21 days, 22 days, 23 days, 24 days, 25 days, 26 days, 27 days, 28 days,29 days, 30 days, 60 days, or longer or any time therebetween. Incertain embodiments, the cellular function is transiently altered, e.g.,such as a modulation that persists for no more than about 30 mins toabout 7 days, or no more than about 1 hr, 2 hrs, 3 hrs, 4 hrs, 5 hrs, 6hrs, 7 hrs, 8 hrs, 9 hrs, 10 hrs, 11 hrs, 12 hrs, 13 hrs, 14 hrs, 15hrs, 16 hrs, 17 hrs, 18 hrs, 19 hrs, 20 hrs, 21 hrs, 22 hrs, 24 hrs, 36hrs, 48 hrs, 60 hrs, 72 hrs, 4 days, 5 days, 6 days, 7 days, or any timetherebetween.

In some embodiments, the circular polyribonucleotide is at least about20 nucleotides, at least about 30 nucleotides, at least about 40nucleotides, at least about 50 nucleotides, at least about 75nucleotides, at least about 100 nucleotides, at least about 200nucleotides, at least about 300 nucleotides, at least about 400nucleotides, at least about 500 nucleotides, at least about 1,000nucleotides, at least about 2,000 nucleotides, at least about 5,000nucleotides, at least about 6,000 nucleotides, at least about 7,000nucleotides, at least about 8,000 nucleotides, at least about 9,000nucleotides, at least about 10,000 nucleotides, at least about 12,000nucleotides, at least about 14,000 nucleotides, at least about 15,000nucleotides, at least about 16,000 nucleotides, at least about 17,000nucleotides, at least about 18,000 nucleotides, at least about 19,000nucleotides, or at least about 20,000 nucleotides. In some embodiments,the circular polyribonucleotide may be of a sufficient size toaccommodate a binding site for a ribosome. One of skill in the art canappreciate that the maximum size of a circular polyribonucleotide can beas large as is within the technical constraints of producing a circularpolyribonucleotide, and/or using the circular polyribonucleotide. Whilenot being bound by theory, it is possible that multiple segments of RNAmay be produced from DNA and their 5′ and 3′ free ends annealed toproduce a “string” of RNA, which ultimately may be circularized whenonly one 5′ and one 3′ free end remains. In some embodiments, themaximum size of a circular polyribonucleotide may be limited by theability of packaging and delivering the RNA to a target. In someembodiments, the size of a circular polyribonucleotide is a lengthsufficient to encode useful polypeptides, and thus, lengths of at least20,000 nucleotides, at least 15,000 nucleotides, at least 10,000nucleotides, at least 7,500 nucleotides, or at least 5,000 nucleotides,at least 4,000 nucleotides, at least 3,000 nucleotides, at least 2,000nucleotides, at least 1,000 nucleotides, at least 500 nucleotides, atleast t 400 nucleotides, at least 300 nucleotides, at least 200nucleotides, at least 100 nucleotides may be useful.

In some embodiments, the circular polyribonucleotide comprises one ormore elements described elsewhere herein. In some embodiments, theelements may be separated from one another by a spacer sequence orlinker. In some embodiments, the elements may be separated from oneanother by 1 ribonucleotide, 2 nucleotides, about 5 nucleotides, about10 nucleotides, about 15 nucleotides, about 20 nucleotides, about 30nucleotides, about 40 nucleotides, about 50 nucleotides, about 60nucleotides, about 80 nucleotides, about 100 nucleotides, about 150nucleotides, about 200 nucleotides, about 250 nucleotides, about 300nucleotides, about 400 nucleotides, about 500 nucleotides, about 600nucleotides, about 700 nucleotides, about 800 nucleotides, about 900nucleotides, about 1000 nucleotides, up to about 1 kb, at least about1000 nucleotides, any amount of nucleotides therebetween. In someembodiments, one or more elements are contiguous with one another, e.g.,lacking a spacer element. In some embodiments, one or more elements inthe circular polyribonucleotide is conformationally flexible. In someembodiments, the conformational flexibility is due to the sequence beingsubstantially free of a secondary structure. In some embodiments, thecircular polyribonucleotide comprises a secondary or tertiary structurethat accommodates one or more desired functions or characteristicsdescribed herein, e.g., accommodate a binding site for a ribosome, e.g.,translation, e.g., rolling circle translation.

In some embodiments, the circular polyribonucleotide comprisesparticular sequence characteristics. For example, the circularpolyribonucleotide may comprise a particular nucleotide composition. Insome such embodiments, the circular polyribonucleotide may include oneor more purine rich regions (adenine or guanosine). In some suchembodiments, the circular polyribonucleotide may include one or morepurine rich regions (adenine or guanosine). In some embodiments, thecircular polyribonucleotide may include one or more AU rich regions orelements (AREs). In some embodiments, the circular polyribonucleotidemay include one or more adenine rich regions.

In some embodiments, the circular polyribonucleotide may include one ormore repetitive elements described elsewhere herein.

In some embodiments, the circular polyribonucleotide comprises one ormore modifications described elsewhere herein.

In some embodiments, the circular polyribonucleotide comprises one ormore expression sequences and is configured for persistent expression ina cell of a subject in vivo. In some embodiments, the circularpolyribonucleotide is configured such that expression of the one or moreexpression sequences in the cell at a later time point is equal to orhigher than an earlier time point. In such embodiments, the expressionof the one or more expression sequences can be either maintained at arelatively stable level or can increase over time. The expression of theexpression sequences can be relatively stable for an extended period oftime. For instance, in some cases, the expression of the one or moreexpression sequences in the cell over a time period of at least 7, 8, 9,10, 12, 14, 16, 18, 20, 22, 23 or more days does not decrease by 50%,45%, 40%, 35%, 30%, 25%, 20%, 15%, 10%, or 5%. In some cases, in somecases, the expression of the one or more expression sequences in thecell is maintained at a level that does not vary by more than 50%, 45%,40%, 35%, 30%, 25%, 20%, 15%, 10%, or 5% for at least 7, 8, 9, 10, 12,14, 16, 18, 20, 22, 23 or more days.

Expression Sequences

Peptides or Polypeptides

In some embodiments, the circular polyribonucleotide comprises at leastone expression sequence that encodes a peptide or polypeptide. Suchpeptide may include, but is not limited to, small peptide,peptidomimetic (e.g., peptoid), amino acids, and amino acid analogs. Thepeptide may be linear or branched. Such peptide may have a molecularweight less than about 5,000 grams per mole, a molecular weight lessthan about 2,000 grams per mole, a molecular weight less than about1,000 grams per mole, a molecular weight less than about 500 grams permole, and salts, esters, and other pharmaceutically acceptable forms ofsuch compounds. Such peptide may include, but is not limited to, aneurotransmitter, a hormone, a drug, a toxin, a viral or microbialparticle, a synthetic molecule, and agonists or antagonists thereof.

The polypeptide may be linear or branched. The polypeptide may have alength from about 5 to about 40,000 amino acids, about 15 to about35,000 amino acids, about 20 to about 30,000 amino acids, about 25 toabout 25,000 amino acids, about 50 to about 20,000 amino acids, about100 to about 15,000 amino acids, about 200 to about 10,000 amino acids,about 500 to about 5,000 amino acids, about 1,000 to about 2,500 aminoacids, or any range therebetween. In some embodiments, the polypeptidehas a length of less than about 40,000 amino acids, less than about35,000 amino acids, less than about 30,000 amino acids, less than about25,000 amino acids, less than about 20,000 amino acids, less than about15,000 amino acids, less than about 10,000 amino acids, less than about9,000 amino acids, less than about 8,000 amino acids, less than about7,000 amino acids, less than about 6,000 amino acids, less than about5,000 amino acids, less than about 4,000 amino acids, less than about3,000 amino acids, less than about 2,500 amino acids, less than about2,000 amino acids, less than about 1,500 amino acids, less than about1,000 amino acids, less than about 900 amino acids, less than about 800amino acids, less than about 700 amino acids, less than about 600 aminoacids, less than about 500 amino acids, less than about 400 amino acids,less than about 300 amino acids, or less may be useful.

Some examples of a peptide or polypeptide include, but are not limitedto, fluorescent tag or marker, antigen, peptide therapeutic, syntheticor analog peptide from naturally-bioactive peptide, agonist orantagonist peptide, anti-microbial peptide, pore-forming peptide, abicyclic peptide, a targeting or cytotoxic peptide, a degradation orself-destruction peptide, and degradation or self-destruction peptides.Peptides useful in the invention described herein also includeantigen-binding peptides, e.g., antigen binding antibody orantibody-like fragments, such as single chain antibodies, nanobodies(see, e.g., Steeland et al. 2016. Nanobodies as therapeutics: bigopportunities for small antibodies. Drug Discov Today: 21(7):1076-113).Such antigen binding peptides may bind a cytosolic antigen, a nuclearantigen, an intra-organellar antigen.

In some embodiments, the circular polyribonucleotide comprises one ormore RNA expression sequences, each of which may encode a polypeptide.The polypeptide may be produced in substantial amounts. As such, thepolypeptide may be any proteinaceous molecule that can be produced. Apolypeptide can be a polypeptide that can be secreted from a cell, orlocalized to the cytoplasm, nucleus or membrane compartment of a cell.Some polypeptides include, but are not limited to, at least a portion ofa viral envelope protein, metabolic regulatory enzymes (e.g., thatregulate lipid or steroid production), an antigen, a toleragen, acytokine, a toxin, enzymes whose absence is associated with a disease,and polypeptides that are not active in an animal until cleaved (e.g.,in the gut of an animal), and a hormone.

In some embodiments, the circular polyribonucleotide includes anexpression sequence encoding a protein e.g., a therapeutic protein. Insome embodiments, therapeutic proteins that can be expressed from thecircular polyribonucleotide disclosed herein have antioxidant activity,binding, cargo receptor activity, catalytic activity, molecular carrieractivity, molecular function regulator, molecular transducer activity,nutrient reservoir activity, protein tag, structural molecule activity,toxin activity, transcription regulator activity, translation regulatoractivity, or transporter activity. Some examples of therapeutic proteinsmay include, but are not limited to, an enzyme replacement protein, aprotein for supplementation, a protein vaccination, antigens (e.g. tumorantigens, viral, bacterial), hormones, cytokines, antibodies,immunotherapy (e.g. cancer), cellular reprogramming/transdifferentiationfactor, transcription factors, chimeric antigen receptor, transposase ornuclease, immune effector (e.g., influences susceptibility to an immuneresponse/signal), a regulated death effector protein (e.g., an inducerof apoptosis or necrosis), a non-lytic inhibitor of a tumor (e.g., aninhibitor of an oncoprotein), an epigenetic modifying agent, epigeneticenzyme, a transcription factor, a DNA or protein modification enzyme, aDNA-intercalating agent, an efflux pump inhibitor, a nuclear receptoractivator or inhibitor, a proteasome inhibitor, a competitive inhibitorfor an enzyme, a protein synthesis effector or inhibitor, a nuclease, aprotein fragment or domain, a ligand or a receptor, and a CRISPR systemor component thereof.

In some embodiments, exemplary proteins that can be expressed from thecircular polyribonucleotide disclosed herein include human proteins, forinstance, receptor binding protein, hormone, growth factor, growthfactor receptor modulator, and regenerative protein (e.g., proteinsimplicated in proliferation and differentiation, e.g., therapeuticprotein, for wound healing). In some embodiments, exemplary proteinsthat can be expressed from the circular polyribonucleotide disclosedherein include EGF (epithelial growth factor). In some embodiments,exemplary proteins that can be expressed from the circularpolyribonucleotide disclosed herein include enzymes, for instance,oxidoreductase enzymes, metabolic enzymes, mitochondrial enzymes,oxygenases, dehydrogenases, ATP-independent enzyme, and desaturases. Insome embodiments, exemplary proteins that can be expressed from thecircular polyribonucleotide disclosed herein include an intracellularprotein or cytosolic protein. In some embodiments, the circularpolyribonucleotide expresses a NanoLuc® luciferase (nLuc). In someembodiments, exemplary proteins that can be expressed from the circularpolyribonucleotide disclosed herein include a secretary protein, forinstance, a secretary enzyme. In some cases, the circularpolyribonucleotide expresses a secretary protein that can have a shorthalf-life therapeutic in the blood, or can be a protein with asubcellular localization signal, or protein with secretory signalpeptide. In some embodiments, the circular polyribonucleotide expressesa Gaussia Luciferase (gLuc). In some cases, the circularpolyribonucleotide expresses a non-human protein, for instance, afluorescent protein, an energy-transfer acceptor, or a protein-tag likeFlag, Myc, or His. In some embodiments, exemplary proteins that can beexpressed from the circular polyribonucleotide include a GFP. In someembodiments, the circular polyribonucleotide expresses tagged proteins,e.g., fusion proteins or engineered proteins containing a protein tage,e.g., chitin binding protein (CBP), maltose binding protein (MBP), Fctag, glutathione-S-transferase (GST), AviTag (GLNDIFEAQKIEWHE (SEQ IDNO: 81)), Calmodulin-tag (KRRWKKNFIAVSAANRFKKISSSGAL (SEQ ID NO: 82));polyglutamate tag (EEEEEE (SEQ ID NO: 83)); E-tag (GAPVPYPDPLEPR (SEQ IDNO: 84)); FLAG-tag (DYKDDDDK (SEQ ID NO: 85)), HA-tag (YPYDVPDYA (SEQ IDNO: 86)); His-tag (HHHHHH (SEQ ID NO: 87)); Myc-tag (EQKLISEEDL (SEQ IDNO: 88)); NE-tag (TKENPRSNQEESYDDNES (SEQ ID NO: 89)); S-tag(KETAAAKFERQHMDS (SEQ ID NO: 90)); SBP-tag(MDEKTTGWRGGHVVEGLAGELEQLRARLEHHPQGQREP (SEQ ID NO: 91)); Softag 1(SLAELLNAGLGGS (SEQ ID NO: 92)); Softag 3 (TQDPSRVG (SEQ ID NO: 93));Spot-tag (PDRVRAVSHWSS (SEQ ID NO: 94)); Strep-tag (Strep-tag II:WSHPQFEK (SEQ ID NO: 95)); TC tag (CCPGCC (SEQ ID NO: 96)); Ty tag(EVHTNQDPLD (SEQ ID NO: 97)); V5 tag (GKPIPNPLLGLDST (SEQ ID NO: 98));VSV-tag (YTDIEMNRLGK (SEQ ID NO: 99)); or Xpress tag (DLYDDDDK (SEQ IDNO: 100)).

In some embodiments, the circular polyribonucleotide expresses anantibody, e.g., an antibody fragment, or a portion thereof. In someembodiments, the antibody expressed by the circular polyribonucleotidecan be of any isotype, such as IgA, IgD, IgE, IgG, IgM. In someembodiments, the circular polyribonucleotide expresses a portion of anantibody, such as a light chain, a heavy chain, a Fc fragment, a CDR(complementary determining region), a Fv fragment, or a Fab fragment, afurther portion thereof. In some embodiments, the circularpolyribonucleotide expresses one or more portions of an antibody. Forinstance, the circular polyribonucleotide can comprise more than oneexpression sequence, each of which expresses a portion of an antibody,and the sum of which can constitute the antibody. In some cases, thecircular polyribonucleotide comprises one expression sequence coding forthe heavy chain of an antibody, and another expression sequence codingfor the light chain of the antibody. In some cases, when the circularpolyribonucleotide is expressed in a cell or a a cell-free environment,the light chain and heavy chain can be subject to appropriatemodification, folding, or other post-translation modification to form afunctional antibody.

Regulatory Elements

In some embodiments, the circular polyribonucleotide comprises aregulatory element, e.g., a sequence that modifies expression of anexpression sequence within the circular polyribonucleotide.

A regulatory element may include a sequence that is located adjacent toan expression sequence that encodes an expression product. A regulatoryelement may be linked operatively to the adjacent sequence. A regulatoryelement may increase an amount of product expressed as compared to anamount of the expressed product when no regulatory element exists. Inaddition, one regulatory element can increase an amount of productsexpressed for multiple expression sequences attached in tandem. Hence,one regulatory element can enhance the expression of one or moreexpression sequences. Multiple regulatory element are well-known topersons of ordinary skill in the art.

A regulatory element as provided herein can include a selectivetranslation sequence. As used herein, the term “selective translationsequence” can refer to a nucleic acid sequence that selectivelyinitiates or activates translation of an expression sequence in thecircular polyribonucleotide, for instance, certain riboswtich aptazymes.A regulatory element can also include a selective degradation sequence.As used herein, the term “selective degradation sequence” can refer to anucleic acid sequence that initiates degradation of the circularpolyribonucleotide, or an expression product of the circularpolyribonucleotide. Exemplary selective degradation sequence can includeriboswitch aptazymes and miRNA binding sites.

In some embodiments, the regulatory element is a translation modulator.A translation modulator can modulate translation of the expressionsequence in the circular polyribonucleotide. A translation modulator canbe a translation enhancer or suppressor. In some embodiments, thecircular polyribonucleotide includes at least one translation modulatoradjacent to at least one expression sequence. In some embodiments, thecircular polyribonucleotide includes a translation modulator adjacenteach expression sequence. In some embodiments, the translation modulatoris present on one or both sides of each expression sequence, leading toseparation of the expression products, e.g., peptide(s) and orpolypeptide(s).

In some embodiments, a translation initiation sequence can function as aregulatory element. In some embodiments, a translation initiationsequence comprises an AUG codon. In some embodiments, a translationinitiation sequence comprises any eukaryotic start codon such as AUG,CUG, GUG, UUG, ACG, AUC, AUU, AAG, AUA, or AGG. In some embodiments, atranslation initiation sequence comprises a Kozak sequence. In someembodiments, translation begins at an alternative translation initiationsequence, e.g., translation initiation sequence other than AUG codon,under selective conditions, e.g., stress induced conditions. As anon-limiting example, the translation of the circular polyribonucleotidemay begin at alternative translation initiation sequence, such as ACG.As another non-limiting example, the circular polyribonucleotidetranslation may begin at alternative translation initiation sequence,CTG/CUG. As yet another non-limiting example, the circularpolyribonucleotide translation may begin at alternative translationinitiation sequence, GTG/GUG. As yet another non-limiting example, thecircular polyribonucleotide may begin translation at a repeat-associatednon-AUG (RAN) sequence, such as an alternative translation initiationsequence that includes short stretches of repetitive RNA e.g. CGG,GGGGCC, CAG, CTG.

Nucleotides flanking a codon that initiates translation, such as, butnot limited to, a start codon or an alternative start codon, are knownto affect the translation efficiency, the length and/or the structure ofthe circular polyribonucleotide. (See e.g., Matsuda and Mauro PLoS ONE,2010 5: 11; the contents of which are herein incorporated by referencein its entirety). Masking any of the nucleotides flanking a codon thatinitiates translation may be used to alter the position of translationinitiation, translation efficiency, length and/or structure of thecircular polyribonucleotide.

In one embodiment, a masking agent may be used near the start codon oralternative start codon in order to mask or hide the codon to reduce theprobability of translation initiation at the masked start codon oralternative start codon. Non-limiting examples of masking agents includeantisense locked nucleic acids (LNA) oligonucleotides and exon junctioncomplexes (EJCs). (See e.g., Matsuda and Mauro describing masking agentsLNA oligonucleotides and EJCs (PLoS ONE, 2010 5: 11); the contents ofwhich are herein incorporated by reference in its entirety). In anotherembodiment, a masking agent may be used to mask a start codon of thecircular polyribonucleotide in order to increase the likelihood thattranslation will initiate at an alternative start codon.

In some embodiments, translation is initiated under selectiveconditions, such as but not limited to viral induced selection in thepresence of GRSF-1 and the circular polyribonucleotide includes GRSF-1binding sites, see for examplehttp://jvi.asm.org/content/76/20/10417.full.

Translation Initiation Sequence

In some embodiments, the circular polyribonucleotide encodes apolypeptide and may comprise a translation initiation sequence, e.g., astart codon. In some embodiments, the translation initiation sequenceincludes a Kozak or Shine-Dalgarno sequence. In some embodiments, thecircular polyribonucleotide includes the translation initiationsequence, e.g., Kozak sequence, adjacent to an expression sequence. Insome embodiments, the translation initiation sequence is a non-codingstart codon. In some embodiments, the translation initiation sequence,e.g., Kozak sequence, is present on one or both sides of each expressionsequence, leading to separation of the expression products. In someembodiments, the circular polyribonucleotide includes at least onetranslation initiation sequence adjacent to an expression sequence. Insome embodiments, the translation initiation sequence providesconformational flexibility to the circular polyribonucleotide. In someembodiments, the translation initiation sequence is within asubstantially single stranded region of the circular polyribonucleotide.

The circular polyribonucleotide may include more than 1 start codon suchas, but not limited to, at least 2, at least 3, at least 4, at least 5,at least 6, at least 7, at least 8, at least 9, at least 10, at least11, at least 12, at least 13, at least 14, at least 15, at least 16, atleast 17, at least 18, at least 19, at least 20, at least 25, at least30, at least 35, at least 40, at least 50, at least 60 or more than 60start codons. Translation may initiate on the first start codon or mayinitiate downstream of the first start codon.

In some embodiments, the circular polyribonucleotide may initiate at acodon which is not the first start codon, e.g., AUG. Translation of thecircular polyribonucleotide may initiate at an alternative translationinitiation sequence, such as, but not limited to, ACG, AGG, AAG,CTG/CUG, GTG/GUG, ATA/AUA, ATT/AUU, TTG/UUG (see Touriol et al. Biologyof the Cell 95 (2003) 169-178 and Matsuda and Mauro PLoS ONE, 2010 5:11; the contents of each of which are herein incorporated by referencein their entireties). In some embodiments, translation begins at analternative translation initiation sequence under selective conditions,e.g., stress induced conditions. As a non-limiting example, thetranslation of the circular polyribonucleotide may begin at alternativetranslation initiation sequence, such as ACG. As another non-limitingexample, the circular polyribonucleotide translation may begin atalternative translation initiation sequence, CTG/CUG. As yet anothernon-limiting example, the circular polyribonucleotide translation maybegin at alternative translation initiation sequence, GTG/GUG. As yetanother non-limiting example, the circular polyribonucleotide may begintranslation at a repeat-associated non-AUG (RAN) sequence, such as analternative translation initiation sequence that includes shortstretches of repetitive RNA e.g. CGG, GGGGCC, CAG, CTG.

In some embodiments, translation is initiated by eukaryotic initiationfactor 4A (eIF4A) treatment with Rocaglates (translation is repressed byblocking 43S scanning, leading to premature, upstream translationinitiation and reduced protein expression from transcripts bearing theRocA-eIF4A target sequence, see for example,www.nature.com/articles/nature17978).

IRES

In some embodiments, the circular polyribonucleotide described hereincomprises an internal ribosome entry site (IRES) element. A suitableIRES element to include in a circular polyribonucleotide comprises anRNA sequence capable of engaging an eukaryotic ribosome. In someembodiments, the IRES element is at least about 5 nt, at least about 8nt, at least about 9 nt, at least about 10 nt, at least about 15 nt, atleast about 20 nt, at least about 25 nt, at least about 30 nt, at leastabout 40 nt, at least about 50 nt, at least about 100 nt, at least about200 nt, at least about 250 nt, at least about 350 nt, or at least about500 nt. In one embodiment, the IRES element is derived from the DNA ofan organism including, but not limited to, a virus, a mammal, and aDrosophila. Such viral DNA may be derived from, but is not limited to,picornavirus complementary DNA (cDNA), with encephalomyocarditis virus(EMCV) cDNA and poliovirus cDNA. In one embodiment, Drosophila DNA fromwhich an IRES element is derived includes, but is not limited to, anAntennapedia gene from Drosophila melanogaster.

In some embodiments, the IRES element is at least partially derived froma virus, for instance, it can be derived from a viral IRES element, suchas ABPV_IGRpred, AEV, ALPV_IGRpred, BQCV_IGRpred, BVDV1_1-385,BVDV1_29-391, CrPV_SNCR, CrPV_IGR, crTMV_IREScp, crTMV_IRESmp75,crTMV_IRESmp228, crTMV_IREScp, crTMV_IREScp, CSFV, CVB3, DCV IGR,EMCV-R, EoPV SNTR, ERAV_245-961, ERBV_162-920, EV71_1-748, FeLV-Notch2,FMDV_type_C, GBV-A, GBV-B, GBV-C, gypsy_env, gypsyD5, gypsyD2,HAV_HM175, HCV_type_1a, HiPV_IGRpred, HIV-1, HoCV1_IGRpred, HRV-2,IAPV_IGRpred, idefix, KBV_IGRpred, LINE-1_ORF1_-101_to_-1,LINE-1_ORF1_-302_to_-202, LINE-1_ORF2_-138to_-86, LINE-1_ORF1_-44to_-1,PSIV_IGR, PV_type1_Mahoney, PV_type3_Leon, REV-A, RhPV_5NCR, RhPV_IGR,SINV1_IGRpred, SV40_661-830, TMEV, TMV_UI_IRESmp228, TRV_5NTR, TrV_IGR,or TSV_IGR. In some embodiments, the IRES element is at least partiallyderived from a cellular IRES, such as AML1/RUNX1, Antp-D, Antp-DE,Antp-CDE, Apaf-1, Apaf-1, AQP4, AT1R_var1, AT1R_var2, AT1R_var3,AT1R_var4, BAG1_p36delta236nt, BAG1_p36, BCL2, BiP_-222_-3,c-IAP1_285-1399, c-IAP1_1313-1462, c-jun, c-myc, Cat-1224, CCND1, DAPS,eIF4G, eIF4GI-ext, eIF4GII, eIF4GII-long, ELG1, ELH, FGF1A, FMR1,Gtx-133-141, Gtx-1-166, Gtx-1-120, Gtx-1-196, hairless, HAP4, HIF1a,hSNM1, Hsp101, hsp70, hsp70, Hsp90, IGF2_leader2, Kv1.4_1.2, L-myc,LamB1_-335-1, MNT_75-267, MNT_36-160, MTG8a, MYB, MYT2_997-1152, n-MYC,NDST1, NDST2, NDST3, NDST4L, NDST4S, NRF_-653_-17, NtHSF1, ODC1,p27kip1, p53_128-269, PDGF2/c-sis, Pim-1, PITSLRE_p58, Rbm3, reaper,Scamper, TFIID, TIF4631, Ubx_1-966, Ubx_373-961, UNR, Ure2, UtrA,VEGF-A_-133_-1, XIAP_5-464, XIAP_305-466, or YAP1. In some embodiments,the IRES element comprises a synthetic IRES, for instance, (GAAA)16 (SEQID NO: 130), (PPT19)4, KMI1, KMI1, KMI2, KMI2, KMIX, X1, or X2.

In some embodiments, the circular polyribonucleotide includes at leastone IRES flanking at least one (e.g., 2, 3, 4, 5 or more) expressionsequence. In some embodiments, the IRES flanks both sides of at leastone (e.g., 2, 3, 4, 5 or more) expression sequence. In some embodiments,the circular polyribonucleotide includes one or more IRES sequences onone or both sides of each expression sequence, leading to separation ofthe resulting peptide(s) and or polypeptide(s).

Termination Element

In some embodiments, the circular polyribonucleotide includes one ormore expression sequences and each expression sequence may or may nothave a termination element. In some embodiments, the circularpolyribonucleotide includes one or more expression sequences and theexpression sequences lack a termination element, such that the circularpolyribonucleotide is continuously translated. Exclusion of atermination element may result in rolling circle translation orcontinuous expression of expression product, e.g., peptides orpolypeptides, due to lack of ribosome stalling or fall-off. In such anembodiment, rolling circle translation expresses a continuous expressionproduct through each expression sequence. In some other embodiments, atermination element of an expression sequence can be part of a staggerelement. In some embodiments, one or more expression sequences in thecircular polyribonucleotide comprises a termination element. However,rolling circle translation or expression of a succeeding (e.g., second,third, fourth, fifth, etc.) expression sequence in the circularpolyribonucleotide is performed. In such instances, the expressionproduct may fall off the ribosome when the ribosome encounters thetermination element, e.g., a stop codon, and terminates translation. Insome embodiments, translation is terminated while the ribosome, e.g., atleast one subunit of the ribosome, remains in contact with the circularpolyribonucleotide.

In some embodiments, the circular polyribonucleotide includes atermination element at the end of one or more expression sequences. Insome embodiments, one or more expression sequences comprises two or moretermination elements in succession. In such embodiments, translation isterminated and rolling circle translation is terminated. In someembodiments, the ribosome completely disengages with the circularpolyribonucleotide. In some such embodiments, production of a succeeding(e.g., second, third, fourth, fifth, etc.) expression sequence in thecircular polyribonucleotide may require the ribosome to reengage withthe circular polyribonucleotide prior to initiation of translation.Generally, termination elements include an in-frame nucleotide tripletthat signals termination of translation, e.g., UAA, UGA, UAG. In someembodiments, one or more termination elements in the circularpolyribonucleotide are frame-shifted termination elements, such as butnot limited to, off-frame or −1 and +1 shifted reading frames (e.g.,hidden stop) that may terminate translation. Frame-shifted terminationelements include nucleotide triples, TAA, TAG, and TGA that appear inthe second and third reading frames of an expression sequence.Frame-shifted termination elements may be important in preventingmisreads of mRNA, which is often detrimental to the cell.

Stagger Element

In some embodiments, the circular polyribonucleotide includes at leastone stagger element adjacent to an expression sequence. In someembodiments, the circular polyribonucleotide includes a stagger elementadjacent to each expression sequence. In some embodiments, the staggerelement is present on one or both sides of each expression sequence,leading to separation of the expression products, e.g., peptide(s) andor polypeptide(s). In some embodiments, the stagger element is a portionof the one or more expression sequences. In some embodiments, thecircular polyribonucleotide comprises one or more expression sequences,and each of the one or more expression sequences is separated from asucceeding expression sequence by a stagger elementon the circularpolyribonucleotide. In some embodiments, the stagger element preventsgeneration of a single polypeptide (a) from two rounds of translation ofa single expression sequence or (b) from one or more rounds oftranslation of two or more expression sequences. In some embodiments,the stagger element is a sequence separate from the one or moreexpression sequences. In some embodiments, the stagger element comprisesa portion of an expression sequence of the one or more expressionsequences.

In some embodiments, the circular polyribonucleotide includes a staggerelement. To avoid production of a continuous expression product, e.g.,peptide or polypeptide, while maintaining rolling circle translation, astagger element may be included to induce ribosomal pausing duringtranslation. In some embodiments, the stagger element is at 3′ end of atleast one of the one or more expression sequences. The stagger elementcan be configured to stall a ribosome during rolling circle translationof the circular polyribonucleotide. The stagger element may include, butis not limited to a 2A-like, or CHYSEL (cis-acting hydrolase element)sequence. In some embodiments, the stagger element encodes a sequencewith a C-terminal consensus sequence that is X₁X₂X₃EX₅NPGP (SEQ ID NO:101), where X1 is absent or G or H, X2 is absent or D or G, X₃ is D or Vor I or S or M, and X₅ is any amino acid. In some embodiments, thissequence comprises a non-conserved sequence of amino-acids with a strongalpha-helical propensity followed by the consensus sequence −D(V/I)ExNPGP (SEQ ID NO: 61), where x=any amino acid. Some nonlimiting examples ofstagger elements includes GDVESNPGP (SEQ ID NO: 62), GDIEENPGP (SEQ IDNO: 63), VEPNPGP (SEQ ID NO: 64), IETNPGP (SEQ ID NO: 65), GDIESNPGP(SEQ ID NO: 66), GDVELNPGP (SEQ ID NO: 67), GDIETNPGP (SEQ ID NO: 68),GDVENPGP (SEQ ID NO: 69), GDVEENPGP (SEQ ID NO: 70), GDVEQNPGP (SEQ IDNO: 71), IESNPGP (SEQ ID NO: 72), GDIELNPGP (SEQ ID NO: 73), HDIETNPGP(SEQ ID NO: 74), HDVETNPGP (SEQ ID NO: 75), HDVEMNPGP (SEQ ID NO: 76),GDMESNPGP (SEQ ID NO: 77), GDVETNPGP (SEQ ID NO: 78), GDIEQNPGP (SEQ IDNO: 79), and DSEFNPGP (SEQ ID NO: 80).

In some embodiments, the stagger element described herein cleaves anexpression product, such as between G and P of the consensus sequencedescribed herein. As one non-limiting example, the circularpolyribonucleotide includes at least one stagger element to cleave theexpression product. In some embodiments, the circular polyribonucleotideincludes a stagger element adjacent to at least one expression sequence.In some embodiments, the circular polyribonucleotide includes a staggerelement after each expression sequence. In some embodiments, thecircular polyribonucleotide includes a stagger element is present on oneor both sides of each expression sequence, leading to translation ofindividual peptide(s) and or polypeptide(s) from each expressionsequence.

In some embodiments, a stagger element comprises one or more modifiednucleotides or unnatural nucleotides that induce ribosomal pausingduring translation. Unnatural nucleotides may include peptide nucleicacid (PNA), Morpholino and locked nucleic acid (LNA), as well as glycolnucleic acid (GNA) and threose nucleic acid (TNA). Examples such asthese are distinguished from naturally occurring DNA or RNA by changesto the backbone of the molecule. Exemplary modifications can include anymodification to the sugar, the nucleobase, the internucleoside linkage(e.g. to a linking phosphate/to a phosphodiester linkage/to thephosphodiester backbone), and any combination thereof that can induceribosomal pausing during translation. Some of the exemplarymodifications provided herein are described elsewhere herein.

In some embodiments, the stagger element is present in the circularpolyribonucleotide in other forms. For example, in some exemplarycircular polyribonucleotides, a stagger element comprises a terminationelement of a first expression sequence in the circularpolyribonucleotide, and a nucleotide spacer sequence that separates thetermination element from a first translation initiation sequence of anexpression succeeding the first expression sequence. In some examples,the first stagger element of the first expression sequence is upstreamof (5′ to) a first translation initiation sequence of the expressionsucceeding the first expression sequence in the circularpolyribonucleotide. In some cases, the first expression sequence and theexpression sequence succeeding the first expression sequence are twoseparate expression sequences in the circular polyribonucleotide. Thedistance between the first stagger element and the first translationinitiation sequence can enable continuous translation of the firstexpression sequence and its succeeding expression sequence. In someembodiments, the first stagger element comprises a termination elementand separates an expression product of the first expression sequencefrom an expression product of its suceeding expression sequences,thereby creating discrete expression products. In some cases, thecircular polyribonucleotide comprising the first stagger elementupstream of the first translation initiation sequence of the succeedingsequence in the circular polyribonucleotide is continuously translated,while a corresponding circular polyribonucleotide comprising a staggerelement of a second expression sequence that is upstream of a secondtranslation initiation sequence of an expression sequence succeeding thesecond expression sequence is not continuously translated. In somecases, there is only one expression sequence in the circularpolyribonucleotide, and the first expression sequence and its suceedingexpression sequence are the same expression sequence. In some exemplarycircular polyribonucleotides, a stagger element comprises a firsttermination element of a first expression sequence in the circularpolyribonucleotide, and a nucleotide spacer sequence that separates thetermination element from a downstream translation initiation sequence.In some such examples, the first stagger element is upstream of (5′ to)a first translation initiation sequence of the first expression sequencein the circular polyribonucleotide. In some cases, the distance betweenthe first stagger element and the first translation initiation sequenceenables continuous translation of the first expression sequence and anysucceeding expression sequences. In some embodiments, the first staggerelement separates one round expression product of the first expressionsequence from the next round expression product of the first expressionsequences, thereby creating discrete expression products. In some cases,the circular polyribonucleotide comprising the first stagger elementupstream of the first translation initiation sequence of the firstexpression sequence in the circular polyribonucleotide is continuouslytranslated, while a corresponding circular polyribonucleotide comprisinga stagger element upstream of a second translation initiation sequenceof a second expression sequence in the corresponding circularpolyribonucleotide is not continuously translated. In some cases, thedistance between the second stagger element and the second translationinitiation sequence is at least 2×, 3×, 4×, 5×, 6×, 7×, 8×, 9×, or 10×greater in the corresponding circular polyribonucleotide than a distancebetween the first stagger element and the first translation initiationin the circular polyribonucleotide. In some cases, the distance betweenthe first stagger element and the first translation initiation is atleast 2 nt, 3 nt, 4 nt, 5 nt, 6 nt, 7 nt, 8 nt, 9 nt, 10 nt, 11 nt, 12nt, 13 nt, 14 nt, 15 nt, 16 nt, 17 nt, 18 nt, 19 nt, 20 nt, 25 nt, 30nt, 35 nt, 40 nt, 45 nt, 50 nt, 55 nt, 60 nt, 65 nt, 70 nt, 75 nt, orgreater. In some embodiments, the distance between the second staggerelement and the second translation initiation is at least 2 nt, 3 nt, 4nt, 5 nt, 6 nt, 7 nt, 8 nt, 9 nt, 10 nt, 11 nt, 12 nt, 13 nt, 14 nt, 15nt, 16 nt, 17 nt, 18 nt, 19 nt, 20 nt, 25 nt, 30 nt, 35 nt, 40 nt, 45nt, 50 nt, 55 nt, 60 nt, 65 nt, 70 nt, 75 nt, or greater than thedistance between the first stagger element and the first translationinitiation. In some embodiments, the circular polyribonucleotidecomprises more than one expression sequence.

Regulatory Nucleic Acids

In some embodiments, the circular polyribonucleotide comprises one ormore expression sequences that encode regulatory nucleic acid, e.g.,that modifies expression of an endogenous gene and/or an exogenous gene.In some embodiments, the expression sequence of a circularpolyribonucleotide as provided herein can comprise a sequence that isantisense to a regulatory nucleic acid like a non-coding RNA, such as,but not limited to, tRNA, lncRNA, miRNA, rRNA, snRNA, microRNA, siRNA,piRNA, snoRNA, snRNA, exRNA, scaRNA, Y RNA, and hnRNA.

In one embodiment, the regulatory nucleic acid targets a host gene. Theregulatory nucleic acids may include, but are not limited to, a nucleicacid that hybridizes to an endogenous gene (e.g., miRNA, siRNA, mRNA,lncRNA, RNA, DNA, an antisense RNA, gRNA as described herein elsewhere),nucleic acid that hybridizes to an exogenous nucleic acid such as aviral DNA or RNA, nucleic acid that hybridizes to an RNA, nucleic acidthat interferes with gene transcription, nucleic acid that interfereswith RNA translation, nucleic acid that stabilizes RNA or destabilizesRNA such as through targeting for degradation, and nucleic acid thatmodulates a DNA or RNA binding factor. In one embodiments, the sequenceis an miRNA. In some embodiments, the regulatory nucleic acid targets asense strand of a host gene. In some embodiments, the regulatory nucleicacid targets an antisense strand of a host gene

In some embodiments, the circular polyribonucleotide comprises aregulatory nucleic acid, such as a guide RNA (gRNA). In someembodiments, the circular polyribonucleotide comprises a guide RNA orencodes the guide RNA. A gRNA short synthetic RNA composed of a“scaffold” sequence necessary for binding to the incomplete effectormoiety and a user-defined ˜20 nucleotide targeting sequence for agenomic target. In practice, guide RNA sequences are generally designedto have a length of between 17-24 nucleotides (e.g., 19, 20, or 21nucleotides) and complementary to the targeted nucleic acid sequence.Custom gRNA generators and algorithms are available commercially for usein the design of effective guide RNAs. Gene editing has also beenachieved using a chimeric “single guide RNA” (“sgRNA”), an engineered(synthetic) single RNA molecule that mimics a naturally occurringcrRNA-tracrRNA complex and contains both a tracrRNA (for binding thenuclease) and at least one crRNA (to guide the nuclease to the sequencetargeted for editing). Chemically modified sgRNAs have also beendemonstrated to be effective in genome editing; see, for example, Hendelet al. (2015) Nature Biotechnol., 985-991.

The gRNA may recognize specific DNA sequences (e.g., sequences adjacentto or within a promoter, enhancer, silencer, or repressor of a gene).

In one embodiment, the gRNA is used as part of a CRISPR system for geneediting. For the purposes of gene editing, the circularpolyribonucleotide may be designed to include one or multiple guide RNAsequences corresponding to a desired target DNA sequence; see, forexample, Cong et al. (2013) Science, 339:819-823; Ran et al. (2013)Nature Protocols, 8:2281-2308. At least about 16 or 17 nucleotides ofgRNA sequence are required by Cas9 for DNA cleavage to occur; for Cpf1at least about 16 nucleotides of gRNA sequence is needed to achievedetectable DNA cleavage.

Certain regulatory nucleic acids can inhibit gene expression through thebiological process of RNA interference (RNAi). RNAi molecules compriseRNA or RNA-like structures typically containing 15-50 base pairs (suchas about 18-25 base pairs) and having a nucleobase sequence identical(complementary) or nearly identical (substantially complementary) to acoding sequence in an expressed target gene within the cell. RNAimolecules include, but are not limited to: short interfering RNAs(siRNAs), double-strand RNAs (dsRNA), micro RNAs (miRNAs), short hairpinRNAs (shRNA), meroduplexes, and dicer substrates (U.S. Pat. Nos.8,084,599 8,349,809 and 8,513,207).

In some embodiments, the circular polyribonucleotide comprisesregulatory nucleic acids that are RNA or RNA-like structures typicallybetween about 5-500 base pairs (depending on the specific RNA structure,e.g., miRNA 5-30 bps, lncRNA 200-500 bps) and may have a nucleobasesequence identical (complementary) or nearly identical (substantiallycomplementary) to a coding sequence in an expressed target gene withinthe cell.

Long non-coding RNAs (lncRNA) are defined as non-protein codingtranscripts longer than 100 nucleotides. This somewhat arbitrary limitdistinguishes lncRNAs from small regulatory RNAs such as microRNAs(miRNAs), short interfering RNAs (siRNAs), and other short RNAs. Ingeneral, the majority (˜78%) of lncRNAs are characterized astissue-specific. Divergent lncRNAs that are transcribed in the oppositedirection to nearby protein-coding genes (comprise a significantproportion ˜20% of total lncRNAs in mammalian genomes) may possiblyregulate the transcription of the nearby gene. In one embodiment, thecircular polyribonucleotide provided herein comprises a sense strand ofa lncRNA. In one embodiment, the circular polyribonucleotide providedherein comprises an antisense strand of a lncRNA.

The circular polyribonucleotide may encode a regulatory nucleic acidsubstantially complementary, or fully complementary, to all or afragment of an endogenous gene or gene product (e.g., mRNA). Theregulatory nucleic acids may complement sequences at the boundarybetween introns and exons, in between exons, or adjacent to exon, toprevent the maturation of newly-generated nuclear RNA transcripts ofspecific genes into mRNA for transcription. The regulatory nucleic acidsthat are complementary to specific genes can hybridize with the mRNA forthat gene and prevent its translation. The antisense regulatory nucleicacid can be DNA, RNA, or a derivative or hybrid thereof. In someembodiments, the regulatory nucleic acid comprises a protein-bindingsite that can bind to a protein that participates in regulation ofexpression of an endogenous gene or an exogenous gene.

The length of the circular polyribonucleotide may encode a regulatorynucleic acid that hybridizes to a transcript of interest that is betweenabout 5 to 30 nucleotides, between about 10 to 30 nucleotides, or about11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28,29, 30 or more nucleotides. The degree of identity of the regulatorynucleic acid to the targeted transcript should be at least 75%, at least80%, at least 85%, at least 90%, or at least 95%.

The circular polyribonucleotide may encode a micro RNA (miRNA) moleculeidentical to about 5 to about 25 contiguous nucleotides of a targetgene. In some embodiments, the miRNA sequence targets a mRNA andcommences with the dinucleotide AA, comprises a GC-content of about30-70% (about 30-60%, about 40-60%, or about 45%-55%), and does not havea high percentage identity to any nucleotide sequence other than thetarget in the genome of the mammal in which it is to be introduced, forexample as determined by standard BLAST search.

In some embodiments, the circular polyribonucleotide comprises at leastone miRNA, e.g., 2, 3, 4, 5, 6, or more. In some embodiments, thecircular polyribonucleotide comprises a sequence that encodes an miRNAat least about 75%, 80%, 85%, 90% 95%, 96%, 97%, 98%, 99% or 100%nucleotide sequence identity to any one of the nucleotide sequences or asequence that is complementary to a target sequence.

siRNAs and shRNAs resemble intermediates in the processing pathway ofthe endogenous microRNA (miRNA) genes (Bartel, Cell 116:281-297, 2004).In some embodiments, siRNAs can function as miRNAs and vice versa (Zenget al., Mol Cell 9:1327-1333, 2002; Doench et al., Genes Dev 17:438-442,2003). MicroRNAs, like siRNAs, use RISC to downregulate target genes,but unlike siRNAs, most animal miRNAs do not cleave the mRNA. Instead,miRNAs reduce protein output through translational suppression or polyAremoval and mRNA degradation (Wu et al., Proc Natl Acad Sci USA103:4034-4039, 2006). Known miRNA binding sites are within mRNA 3′ UTRs;miRNAs seem to target sites with near-perfect complementarity tonucleotides 2-8 from the miRNA's 5′ end (Rajewsky, Nat Genet 38Suppl:S8-13, 2006; Lim et al., Nature 433:769-773, 2005). This region isknown as the seed region. Because siRNAs and miRNAs are interchangeable,exogenous siRNAs downregulate mRNAs with seed complementarity to thesiRNA (Birmingham et al., Nat Methods 3:199-204, 2006. Multiple targetsites within a 3′ UTR give stronger downregulation (Doench et al., GenesDev 17:438-442, 2003).

Lists of known miRNA sequences can be found in databases maintained byresearch organizations, such as Wellcome Trust Sanger Institute, PennCenter for Bioinformatics, Memorial Sloan Kettering Cancer Center, andEuropean Molecule Biology Laboratory, among others. Known effectivesiRNA sequences and cognate binding sites are also well represented inthe relevant literature. RNAi molecules are readily designed andproduced by technologies known in the art. In addition, there arecomputational tools that increase the chance of finding effective andspecific sequence motifs (Lagana et al., Methods Mol. Bio., 2015,1269:393-412).

The circular polyribonucleotide may modulate expression of RNA encodedby a gene. Because multiple genes can share some degree of sequencehomology with each other, in some embodiments, the circularpolyribonucleotide can be designed to target a class of genes withsufficient sequence homology. In some embodiments, the circularpolyribonucleotide can contain a sequence that has complementarity tosequences that are shared amongst different gene targets or are uniquefor a specific gene target. In some embodiments, the circularpolyribonucleotide can be designed to target conserved regions of an RNAsequence having homology between several genes thereby targeting severalgenes in a gene family (e.g., different gene isoforms, splice variants,mutant genes, etc.). In some embodiments, the circularpolyribonucleotide can be designed to target a sequence that is uniqueto a specific RNA sequence of a single gene.

In some embodiments, the expression sequence has a length less than 5000bps (e.g., less than about 5000 bps, 4000 bps, 3000 bps, 2000 bps, 1000bps, 900 bps, 800 bps, 700 bps, 600 bps, 500 bps, 400 bps, 300 bps, 200bps, 100 bps, 50 bps, 40 bps, 30 bps, 20 bps, 10 bps, or less). In someembodiments, the expression sequence has, independently or in additionto, a length greater than 10 bps (e.g., at least about 10 bps, 20 bps,30 bps, 40 bps, 50 bps, 60 bps, 70 bps, 80 bps, 90 bps, 100 bps, 200bps, 300 bps, 400 bps, 500 bps, 600 bps, 700 bps, 800 bps, 900 bps, 1000kb, 1.1 kb, 1.2 kb, 1.3 kb, 1.4 kb, 1.5 kb, 1.6 kb, 1.7 kb, 1.8 kb, 1.9kb, 2 kb, 2.1 kb, 2.2 kb, 2.3 kb, 2.4 kb, 2.5 kb, 2.6 kb, 2.7 kb, 2.8kb, 2.9 kb, 3 kb, 3.1 kb, 3.2 kb, 3.3 kb, 3.4 kb, 3.5 kb, 3.6 kb, 3.7kb, 3.8 kb, 3.9 kb, 4 kb, 4.1 kb, 4.2 kb, 4.3 kb, 4.4 kb, 4.5 kb, 4.6kb, 4.7 kb, 4.8 kb, 4.9 kb, 5 kb or greater).

In some embodiments, the expression sequence comprises one or more ofthe features described herein, e.g., a sequence encoding one or morepeptides or proteins, one or more regulatory element, one or moreregulatory nucleic acids, e.g., one or more non-coding RNAs, otherexpression sequences, and any combination thereof.

Translation Efficiency

In some embodiments, the translation efficiency of a circularpolyribonucleotide as provided herein is greater than a reference, e.g.,a linear counterpart, a linear expression sequence, or a linear circularpolyribonucleotide. In some embodiments, a circular polyribonucleotideas provided herein has the translation efficiency that is at least about5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%,75%, 80%, 85%, 90%, 95%, 100%, 125%, 150%, 175%, 200%, 250%, 300%, 350%,400%, 450%, 500%, 600%, 70%, 800%, 900%, 1000%, 2000%, 5000%, 10000%,100000%, or more greater than that of a reference. In some embodiments,a circular polyribonucleotide has a translation efficiency 10% greaterthan that of a linear counterpart. In some embodiments, a circularpolyribonucleotide has a translation efficiency 300% greater than thatof a linear counterpart.

In some embodiments, the circular polyribonucleotide producesstoichiometric ratios of expression products. Rolling circle translationcontinuously produces expression products at substantially equivalentratios. In some embodiments, the circular polyribonucleotide has astoichiometric translation efficiency, such that expression products areproduced at substantially equivalent ratios. In some embodiments, thecircular polyribonucleotide has a stoichiometric translation efficiencyof multiple expression products, e.g., products from 2, 3, 4, 5, 6, 7,8, 9, 10, 11, 12, or more expression sequences.

Rolling Circle Translation

In some embodiments, once translation of the circular polyribonucleotideis initiated, the ribosome bound to the circular polyribonucleotide doesnot disengage from the circular polyribonucleotide before finishing atleast one round of translation of the circular polyribonucleotide. Insome embodiments, the circular polyribonucleotide as described herein iscompetent for rolling circle translation. In some embodiments, duringrolling circle translation, once translation of the circularpolyribonucleotide is initiated, the ribosome bound to the circularpolyribonucleotide does not disengage from the circularpolyribonucleotide before finishing at least 2 rounds, at least 3rounds, at least 4 rounds, at least 5 rounds, at least 6 rounds, atleast 7 rounds, at least 8 rounds, at least 9 rounds, at least 10rounds, at least 11 rounds, at least 12 rounds, at least 13 rounds, atleast 14 rounds, at least 15 rounds, at least 20 rounds, at least 30rounds, at least 40 rounds, at least 50 rounds, at least 60 rounds, atleast 70 rounds, at least 80 rounds, at least 90 rounds, at least 100rounds, at least 150 rounds, at least 200 rounds, at least 250 rounds,at least 500 rounds, at least 1000 rounds, at least 1500 rounds, atleast 2000 rounds, at least 5000 rounds, at least 10000 rounds, at least10⁵ rounds, or at least 10⁶ rounds of translation of the circularpolyribonucleotide.

In some embodiments, the rolling circle translation of the circularpolyribonucleotide leads to generation of polypeptide product that istranslated from more than one round of translation of the circularpolyribonucleotide (“continuous” expression product). In someembodiments, the circular polyribonucleotide comprises a staggerelement, and rolling circle translation of the circularpolyribonucleotide leads to generation of polypeptide product that isgenerated from a single round of translation or less than a single roundof translation of the circular polyribonucleotide (“discrete” expressionproduct). In some embodiments, the circular polyribonucleotide isconfigured such that at least 10%, 20%, 30%, 40%, 50%, at least 60%, atleast 70%, at least 80%, at least 90%, at least 95%, at least 96%, atleast 97%, at least 98%, at least 99%, or 100% of total polypeptides(molar/molar) generated during the rolling circle translation of thecircular polyribonucleotide are discrete polypeptides. In someembodiments, the amount ratio of the discrete products over the totalpolypeptides is tested in an in vitro translation system. In someembodiments, the in vitro translation system used for the test of amountratio comprises rabbit reticulocyte lysate. In some embodiments, theamount ratio is tested in an in vivo translation system, such as aeukaryotic cell or a prokaryotic cell, a cultured cell or a cell in anorganism.

Untranslated Regions

In some embodiments, the circular polyribonucleotide comprisesuntranslated regions (UTRs). UTRs of a genomic region comprising a genemay be transcribed but not translated. In some embodiments, a UTR may beincluded upstream of the translation initiation sequence of anexpression sequence described herein. In some embodiments, a UTR may beincluded downstream of an expression sequence described herein. In someinstances, one UTR for first expression sequence is the same as orcontinuous with or overlapping with another UTR for a second expressionsequence. In some embodiments, the intron is a human intron. In someembodiments, the intron is a full length human intron, e.g., ZKSCAN1.

In some embodiments, the circular polyribonucleotide comprises a UTRwith one or more stretches of Adenosines and Uridines embedded within.These AU rich signatures are may increase turnover rates of theexpression product.

Introduction, removal or modification of UTR AU rich elements (AREs) maybe useful to modulate the stability or immunogenicity of the circularpolyribonucleotide. When engineering specific circularpolyribonucleotides, one or more copies of an ARE may be introduced tothe circular polyribonucleotide and the copies of an ARE may modulatetranslation and/or production of an expression product. Likewise, AREsmay be identified and removed or engineered into the circularpolyribonucleotide to modulate the intracellular stability and thusaffect translation and production of the resultant protein.

It should be understood that any UTR from any gene may be incorporatedinto the respective flanking regions of the circular polyribonucleotide.As a non-limiting example, the UTR or a fragment thereof which may beincorporated is a UTR listed in US Provisional Application Nos. U.S.61/775,509 and U.S. 61/829,372, or in International Patent ApplicationNo. PCT/US2014/021522; the contents of each of which are hereinincorporated by reference in its entirety. Furthermore, multiplewild-type UTRs of any known gene may be utilized. It is also within thescope of the present invention to provide artificial UTRs which are notvariants of wild type genes. These UTRs or portions thereof may beplaced in the same orientation as in the transcript from which they wereselected or may be altered in orientation or location. Hence a 5′ or 3′UTR may be inverted, shortened, lengthened, made chimeric with one ormore other 5′ UTRs or 3′ UTRs. As used herein, the term “altered” as itrelates to a UTR sequence, means that the UTR has been changed in someway in relation to a reference sequence. For example, a 3′ or 5′ UTR maybe altered relative to a wild type or native UTR by the change inorientation or location as taught above or may be altered by theinclusion of additional nucleotides, deletion of nucleotides, swappingor transposition of nucleotides. Any of these changes producing an“altered” UTR (whether 3′ or 5′) comprise a variant UTR.

In one embodiment, a double, triple or quadruple UTR, such as a 5′ or 3′UTR, may be used. As used herein, a “double” UTR is one in which twocopies of the same UTR are encoded either in series or substantially inseries. For example, a double beta-globin 3′ UTR may be used asdescribed in US Patent publication 20100129877, the contents of whichare incorporated herein by reference in its entirety.

PolyA Sequence

In some embodiments, the circular polyribonucleotide may include apoly-A sequence. In some embodiments, the length of a poly-A sequence isgreater than 10 nucleotides in length. In one embodiment, the poly-Asequence is greater than 15 nucleotides in length (e.g., at least orgreater than about 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 70, 80,90, 100, 120, 140, 160, 180, 200, 250, 300, 350, 400, 450, 500, 600,700, 800, 900, 1,000, 1,100, 1,200, 1,300, 1,400, 1,500, 1,600, 1,700,1,800, 1,900, 2,000, 2,500, and 3,000 nucleotides). In some embodiments,the poly-A sequence is from about 10 to about 3,000 nucleotides (e.g.,from 30 to 50, from 30 to 100, from 30 to 250, from 30 to 500, from 30to 750, from 30 to 1,000, from 30 to 1,500, from 30 to 2,000, from 30 to2,500, from 50 to 100, from 50 to 250, from 50 to 500, from 50 to 750,from 50 to 1,000, from 50 to 1,500, from 50 to 2,000, from 50 to 2,500,from 50 to 3,000, from 100 to 500, from 100 to 750, from 100 to 1,000,from 100 to 1,500, from 100 to 2,000, from 100 to 2,500, from 100 to3,000, from 500 to 750, from 500 to 1,000, from 500 to 1,500, from 500to 2,000, from 500 to 2,500, from 500 to 3,000, from 1,000 to 1,500,from 1,000 to 2,000, from 1,000 to 2,500, from 1,000 to 3,000, from1,500 to 2,000, from 1,500 to 2,500, from 1,500 to 3,000, from 2,000 to3,000, from 2,000 to 2,500, and from 2,500 to 3,000).

In one embodiment, the poly-A sequence is designed relative to thelength of the overall circular polyribonucleotide. This design may bebased on the length of the coding region, the length of a particularfeature or region (such as the first or flanking regions), or based onthe length of the ultimate product expressed from the circularpolyribonucleotide. In this context, the poly-A sequence may be 10, 20,30, 40, 50, 60, 70, 80, 90, or 100% greater in length than the circularpolyribonucleotide or a feature thereof. The poly-A sequence may also bedesigned as a fraction of circular polyribonucleotide to which itbelongs. In this context, the poly-A sequence may be 10, 20, 30, 40, 50,60, 70, 80, or 90% or more of the total length of the construct or thetotal length of the construct minus the poly-A sequence. Further,engineered binding sites and conjugation of circular polyribonucleotidefor Poly-A binding protein may enhance expression.

In one embodiment, the circular polyribonucleotide is designed toinclude a polyA-G quartet. The G-quartet is a cyclic hydrogen bondedarray of four guanine nucleotides that can be formed by G-rich sequencesin both DNA and RNA. In one embodiment, the G-quartet is incorporated atthe end of the poly-A sequence. The resultant circularpolyribonucleotide construct is assayed for stability, proteinproduction and/or other parameters including half-life at various timepoints. In some embodiments, the polyA-G quartet results in proteinproduction equivalent to at least 75% of that seen using a poly-Asequence of 120 nucleotides alone.

In some embodiments, the circular polyribonucleotide comprises a polyA,lacks a polyA, or has a modified polyA to modulate one or morecharacteristics of the circular polyribonucleotide. In some embodiments,the circular polyribonucleotide lacking a polyA or having modified polyAimproves one or more functional characteristics, e.g., immunogenicity,half-life, expression efficiency, etc.

RNA-Binding

In some embodiments, the circular polyribonucleotide comprises one ormore RNA binding sites. microRNAs (or miRNA) are short noncoding RNAsthat bind to the 3′UTR of nucleic acid molecules and down-regulate geneexpression either by reducing nucleic acid molecule stability or byinhibiting translation. The circular polyribonucleotide may comprise oneor more microRNA target sequences, microRNA sequences, or microRNAseeds. Such sequences may correspond to any known microRNA, such asthose taught in US Publication US2005/0261218 and US PublicationUS2005/0059005, the contents of which are incorporated herein byreference in their entirety.

A microRNA sequence comprises a “seed” region, i.e., a sequence in theregion of positions 2-8 of the mature microRNA, which sequence hasperfect Watson-Crick complementarity to the miRNA target sequence. AmicroRNA seed may comprise positions 2-8 or 2-7 of the mature microRNA.In some embodiments, a microRNA seed may comprise 7 nucleotides (e.g.,nucleotides 2-8 of the mature microRNA), wherein the seed-complementarysite in the corresponding miRNA target is flanked by an adenine (A)opposed to microRNA position 1. In some embodiments, a microRNA seed maycomprise 6 nucleotides (e.g., nucleotides 2-7 of the mature microRNA),wherein the seed-complementary site in the corresponding miRNA target isflanked byan adenine (A) opposed to microRNA position 1. See forexample, Grimson A, Farh K, Johnston W K, Garrett-Engele P, Lim L P,Barrel D P; Mol Cell. 2007 Jul. 6; 27(1):91-105; each of which is hereinincorporated by reference in their entirety.

The bases of the microRNA seed are substantially complementary with thetarget sequence. By engineering microRNA target sequences into thecircular polyribonucleotide, the circular polyribonucleotide may evadeor be detected by the host's immune system, have modulated degradation,or modulated translation, provided the microRNA in question isavailable. This process will reduce the hazard of off target effectsupon circular polyribonucleotide delivery. Identification of microRNA,microRNA target regions, and their expression patterns and role inbiology have been reported (Bonauer et al., Curr Drug Targets 201011:943-949; Anand and Cheresh Curr Opin Hematol 2011 18: 171-176;Contreras and Rao Leukemia 2012 26:404-413 (2011 Dec. 20. doi:10.1038/leu.2011.356); Barrel Cell 2009 136:215-233; Landgraf et al,Cell, 2007 129: 1401-1414; each of which is herein incorporated byreference in its entirety).

Conversely, microRNA binding sites can be engineered out of (i.e.removed from) the circular polyribonucleotide to modulate proteinexpression in specific tissues. Regulation of expression in multipletissues can be accomplished through introduction or removal or one orseveral microRNA binding sites.

Examples of tissues where microRNA are known to regulate mRNA, andthereby protein expression, include, but are not limited to, liver(miR-122), muscle (miR-133, miR-206, miR-208), endothelial cells(miR-17-92, miR-126), myeloid cells (miR-142-3p, miR-142-5p, miR-16,miR-21, miR-223, miR-24, miR-27), adipose tissue (let-7, miR-30c), heart(miR-1d, miR-149), kidney (miR-192, miR-194, miR-204), and lungepithelial cells (let-7, miR-133, miR-126). MicroRNA can also regulatecomplex biological processes such as angiogenesis (miR-132) (Anand andCheresh Curr Opin Hematol 2011 18: 171-176; herein incorporated byreference in its entirety). In the circular polyribonucleotide describedherein, binding sites for microRNAs that are involved in such processesmay be removed or introduced, in order to tailor the expression from thecircular polyribonucleotide to biologically relevant cell types or tothe context of relevant biological processes. A listing of MicroRNA, miRsequences and miR binding sites is listed in Table 9 of U.S. ProvisionalApplication No. 61/753,661 filed Jan. 17, 2013, in Table 9 of U.S.Provisional Application No. 61/754,159 filed Jan. 18, 2013, and in Table7 of U.S. Provisional Application No. 61/758,921 filed Jan. 31, 2013,each of which are herein incorporated by reference in their entireties.In some embodiments, the microRNA binding site includes, e.g. miR-7.

The circular polyribonucleotide disclosed herein can comprise a miRNAbinding site that hybridize to any miRNA, such as any of those disclosedin miRNA databases such as miRBase, deepBase, miRBase, microRNA.org,miRGen 2.0; miRNAMap, PMRD, TargetScan, or VIRmiRNA. In some cases, themiRNA binding site can any site that is complementary to an miRNA whosetarget gene is disclosed in microRNA target genedatasese such asStarBase, StarScan, Cupid, TargetScan, TarBase, Diana-microT, miRecords,PicTar, PITA, RepTarm RNA22, miRTarBase, miRwalk, or MBSTAR.

Through an understanding of the expression patterns of microRNA indifferent cell types, the circular polyribonucleotide described hereincan be engineered for more targeted expression in specific cell types oronly under specific biological conditions. Through introduction oftissue-specific microRNA binding sites, the circular polyribonucleotidecan be designed for optimal protein expression in a tissue or in thecontext of a biological condition. Examples of use of microRNA to drivetissue or disease-specific gene expression are listed (Getner andNaldini, Tissue Antigens. 2012, 80:393-403; herein incorporated byreference in its entirety).

In addition, microRNA seed sites may be incorporated into the circularpolyribonucleotide to modulate expression in certain cells which resultsin a biological improvement. An example of this is incorporation ofmiR-142 sites. Incorporation of miR-142 sites into the circularpolyribonucleotide described herein may modulate expression inhematopoietic cells, but also reduce or abolish immune responses to aprotein encoded in the circular polyribonucleotide.

In some embodiments, the circular polyribonucleotide includes one ormore large intergenic non-coding RNAs (lincRNA) binding sites. Largeintergenic non-coding RNAs (lincRNAs) make up most of the longnon-coding RNAs. LincRNAs are non-coding transcripts and, in someembodiments, are more than about 200 nucleotides long. In someembodiments, they have an exon-intron-exon structure, similar toprotein-coding genes, but do not encompass open-reading frames and donot code for proteins. More than 8,000 lincRNAs have been describedrecently and are thought to be the largest subclass of RNAs, originatingfrom the non-coding transcriptome in humans. Thousands of lincRNAs areknown and some appear to be key regulators of diverse cellularprocesses. Determining the function of individual lincRNAs remains achallenge. lincRNA expression is strikingly tissue specific compared tocoding genes, and that they are typically co-expressed with theirneighboring genes, albeit to a similar extent to that of pairs ofneighboring protein-coding genes.

In some embodiments, the circular polyribonucleotide includes one ormore lincRNAs, such as FIRRE, LINC00969, PVT1, LINC01608, JPX,LINC01572, LINC00355, C1orf132, C3orf35, RP11-734, LINC01608,CC-499B15.5, CASC15, LINC00937, RP11-191, etc., or other lincRNAs orlncRNAs such as those from known lncRNA databases.

Protein-Binding

In some embodiments, the circular polyribonucleotide includes one ormore protein binding sites that enable a protein, e.g., a ribosome, tobind to an internal site in the RNA sequence. By engineering proteinbinding sites, e.g., ribosome binding sites, into the circularpolyribonucleotide, the circular polyribonucleotide may evade or havereduced detection by the host's immune system, have modulateddegradation, or modulated translation, by masking the circularpolyribonucleotide from components of the host's immune system.

In some embodiments, the circular polyribonucleotide comprises at leastone immunoprotein binding site, for example to evade immune reponses,e.g., CTL (cytotoxic T lymphocyte) responses. In some embodiments, theimmunoprotein binding site is a nucleotide sequence that binds to animmunoprotein and aids in masking the circular polyribonucleotide asexogenous. In some embodiments, the immunoprotein binding site is anucleotide sequence that binds to an immunoprotein and aids in hidingthe circular polyribonucleotide as exogenous or foreign.

Traditional mechanisms of ribosome engagement to linear RNA involveribosome binding to the capped 5′ end of an RNA. From the 5′ end, theribosome migrates to an initiation codon, whereupon the first peptidebond is formed. According to the present invention, internal initiation(i.e., cap-independent) of translation of the circularpolyribonucleotide does not require a free end or a capped end. Rather,a ribosome binds to a non-capped internal site, whereby the ribosomebegins polypeptide elongation at an initiation codon. In someembodiments, the circular polyribonucleotide includes one or more RNAsequences comprising a ribosome binding site, e.g., an initiation codon.

Natural 5′UTRS bear features which play roles in for translationinitiation. They harbor signatures like Kozak sequences which arecommonly known to be involved in the process by which the ribosomeinitiates translation of many genes. Kozak sequences have the consensusCCR(A/G)CCAUGG, where R is a purine (adenine or guanine) three basesupstream of the start codon (AUG), which is followed by another ‘G’.5′UTR also have been known to form secondary structures which areinvolved in elongation factor binding.

In some embodiments, the circular polyribonucleotide encodes a proteinbinding sequence that binds to a protein. In some embodiments, theprotein binding sequence targets or localizes the circularpolyribonucleotide to a specific target. In some embodiments, theprotein binding sequence specifically binds an arginine-rich region of aprotein.

In some embodiments, the protein binding site includes, but is notlimited to, a binding site to the protein such as ACIN1, AGO, APOBEC3F,APOBEC3G, ATXN2, AUH, BCCIP, CAPRIN1, CELF2, CPSF1, CPSF2, CPSF6, CPSF7,CSTF2, CSTF2T, CTCF, DDX21, DDX3, DDX3X, DDX42, DGCR8, EIF3A, EIF4A3,EIF4G2, ELAVL1, ELAVL3, FAM120A, FBL, FIP1L1, FKBP4, FMR1, FUS, FXR1,FXR2, GNL3, GTF2F1, HNRNPA1, HNRNPA2B1, HNRNPC, HNRNPK, HNRNPL, HNRNPM,HNRNPU, HNRNPUL1, IGF2BP1, IGF2BP2, IGF2BP3, ILF3, KHDRBS1, LARP7,LIN28A, LIN28B, m6A, MBNL2, METTL3, MOV10, MSI1, MSI2, NONO, NONO-,NOP58, NPM1, NUDT21, PCBP2, POLR2A, PRPF8, PTBP1, RBFOX2, RBM10, RBM22,RBM27, RBM47, RNPS1, SAFB2, SBDS, SF3A3, SF3B4, SIRT7, SLBP, SLTM,SMNDC1, SND1, SRRM4, SRSF1, SRSF3, SRSF7, SRSF9, TAF15, TARDBP, TIA1,TNRC6A, TOP3B, TRA2A, TRA2B, U2AF1, U2AF2, UNK, UPF1, WDR33, XRN2, YBX1,YTHDC1, YTHDF1, YTHDF2, YWHAG, ZC3H7B, PDK1, AKT1, and any other proteinthat binds RNA.

Encryptogen

As described herein, the circular polyribonucleotide comprises anencryptogen to reduce, evade or avoid the innate immune response of acell. In one aspect, provided herein are circular polyribonucleotidewhich when delivered to cells, results in a reduced immune response fromthe host as compared to the response triggered by a reference compound,e.g. a linear polynucleotide corresponding to the described circularpolyribonucleotide or a circular polyribonucleotide lacking anencryptogen. In some embodiments, the circular polyribonucleotide hasless immunogenicity than a counterpart lacking an encryptogen.

In some embodiments, an encryptogen enhances stability. There is growingbody of evidence about the regulatory roles played by the UTRs in termsof stability of a nucleic acid molecule and translation. The regulatoryfeatures of a UTR may be included in the encryptogen to enhance thestability of the circular polyribonucleotide.

In some embodiments, 5′ or 3′UTRs can constitute encryptogens in acircular polyribonucleotide. For example, removal or modification of UTRAU rich elements (AREs) may be useful to modulate the stability orimmunogenicity of the circular polyribonucleotide.

In some embodiments, removal of modification of AU rich elements (AREs)in expression sequence, e.g., translatable regions, can be useful tomodulate the stability or immunogenicity of the circularpolyribonucleotide

In some embodiments, an encryptogen comprises miRNA binding site orbinding site to any other non-coding RNAs. For example, incorporation ofmiR-142 sites into the circular polyribonucleotide described herein maynot only modulate expression in hematopoietic cells, but also reduce orabolish immune responses to a protein encoded in the circularpolyribonucleotide.

In some embodiments, an encyptogen comprises one or more protein bindingsites that enable a protein, e.g., an immunoprotein, to bind to the RNAsequence. By engineering protein binding sites into the circularpolyribonucleotide, the circular polyribonucleotide may evade or havereduced detection by the host's immune system, have modulateddegradation, or modulated translation, by masking the circularpolyribonucleotide from components of the host's immune system. In someembodiments, the circular polyribonucleotide comprises at least oneimmunoprotein binding site, for example to evade immune reponses, e.g.,CTL responses. In some embodiments, the immunoprotein binding site is anucleotide sequence that binds to an immunoprotein and aids in maskingthe circular polyribonucleotide as exogenous.

In some embodiments, an encryptogen comprises one or more modifiednucleotides. Exemplary modifications can include any modification to thesugar, the nucleobase, the internucleoside linkage (e.g. to a linkingphosphate/to a phosphodiester linkage/to the phosphodiester backbone),and any combination thereof that can prevent or reduce immune responseagainst the circular polyribonucleotide. Some of the exemplarymodifications provided herein are described in details below.

In some embodiments, the circular polyribonucleotide includes one ormore modifications as described elsewhere herein to reduce an immuneresponse from the host as compared to the response triggered by areference compound, e.g. a circular polyribonucleotide lacking themodifications. In particular, the addition of one or more inosine hasbeen shown to discriminate RNA as endogenous versus viral. See forexample, Yu, Z. et al. (2015) RNA editing by ADAR1 marks dsRNA as“self”. Cell Res. 25, 1283-1284, which is incorporated by reference inits entirety.

In some embodiments, the circular polyribonucleotide includes one ormore expression sequences for shRNA or an RNA sequence that can beprocessed into siRNA, and the shRNA or siRNA targets RIG-1 and reducesexpression of RIG-1. RIG-1 can sense foreign circular RNA and leads todegradation of foreign circular RNA. Therefore, a circularpolynucleotide harboring sequences for RIG-1-targeting shRNA, siRNA orany other regulatory nucleic acids can reduce immunity, e.g., host cellimmunity, against the circular polyribonucleotide.

In some embodiments, the circular polyribonucleotide lacks a sequence,element or structure, that aids the circular polyribonucleotide inreducing, evading or avoiding an innate immune response of a cell. Insome such embodiments, the circular polyribonucleotide may lack a polyAsequence, a 5′ end, a 3′ end, phosphate group, hydroxyl group, or anycombination thereof.

Riboswitches

In some embodiments, the circular polyribonucleotide comprises one ormore riboswitches.

A riboswitch is typically considered a part of the circularpolyribonucleotide that can directly bind a small target molecule, andwhose binding of the target affects RNA translation, the expressionproduct stability and activity (Tucker B J, Breaker R R (2005), CurrOpin Struct Biol 15 (3): 342-8). Thus, the circular polyribonucleotidethat includes a riboswitch is directly involved in regulating its ownactivity, depending on the presence or absence of its target molecule.In some embodiments, a riboswitch has a region of aptamer-like affinityfor a separate molecule. Thus, in the broader context of the instantinvention, any aptamer included within a non-coding nucleic acid couldbe used for sequestration of molecules from bulk volumes. Downstreamreporting of the event via “(ribo)switch” activity may be especiallyadvantageous.

In some embodiments, the riboswitch may have an effect on geneexpression including, but not limited to, transcriptional termination,inhibition of translation initiation, mRNA self-cleavage, and ineukaryotes, alteration of splicing pathways. The riboswitch may functionto control gene expression through the binding or removal of a triggermolecule. Thus, subjecting a circular polyribonucleotide that includesthe riboswitch to conditions that activate, deactivate or block theriboswitch to alter expression. Expression can be altered as a resultof, for example, termination of transcription or blocking of ribosomebinding to the RNA. Binding of a trigger molecule or an analog thereofcan, depending on the nature of the riboswitch, reduce or preventexpression of the RNA molecule or promote or increase expression of theRNA molecule. Some examples of riboswitches are described herein.

In some embodiments, the riboswitch is a Cobalamin riboswitch (alsoB12-element), which binds adenosylcobalamin (the coenzyme form ofvitamin B12) to regulate the biosynthesis and transport of cobalamin andsimilar metabolites.

In some embodiments, the riboswitch is a cyclic di-GMP riboswitches,which bind cyclic di-GMP to regulate a variety of genes. Twonon-structurally related classes exist—cyclic di-GMP-1 and cyclicdi-GMP-11.

In some embodiments, the riboswitch is a FMN riboswitch (alsoRFN-element) which binds flavin mononucleotide (FMN) to regulateriboflavin biosynthesis and transport.

In some embodiments, the riboswitch is a glmS riboswitch, which cleavesitself when there is a sufficient concentration ofglucosamine-6-phosphate.

In some embodiments, the riboswitch is a Glutamine riboswitches, whichbind glutamine to regulate genes involved in glutamine and nitrogenmetabolism. They also bind short peptides of unknown function. Suchriboswitches fall into two classes, which are structurally related: theglnA RNA motif and Downstream-peptide motif.

In some embodiments, the riboswitch is a Glycine riboswitch, which bindsglycine to regulate glycine metabolism genes. It comprises two adjacentaptamer domains in the same mRNA, and is the only known natural RNA thatexhibits cooperative binding.

In some embodiments, the riboswitch is a Lysine riboswitch (also L-box),which binds lysine to regulate lysine biosynthesis, catabolism andtransport.

In some embodiments, the riboswitch is a PreQ1 riboswitch, which bindspre-queuosine to regulate genes involved in the synthesis or transportof this precursor to queuosine. Two entirely distinct classes of PreGIriboswitches are known: PreQ1-1 riboswitches and PreQ1-11 riboswitches.The binding domain of PreQ1-1 riboswitches is unusually small amongnaturally occurring riboswitches. PreGI-II riboswitches, which are onlyfound in certain species in the genera Streptococcus and Lactococcus,have a completely different structure, and are larger.

In some embodiments, the riboswitch is a Purine riboswitch, which bindspurines to regulate purine metabolism and transport. Different forms ofthe purine riboswitch bind guanine (a form originally known as theG-box) or adenine. The specificity for either guanine or adenine dependscompletely upon Watson-Crick interactions with a single pyrimidine inthe riboswitch at position Y74. In the guanine riboswitch, this residueis a cytosine (i.e. C74), in the adenine residue it is always a uracil(i.e. U74). Homologous types of purine riboswitches bind deoxyguanosine,but have more significant differences than a single nucleotide mutation.

In some embodiments, the riboswitch is a SAH riboswitch, which bindsS-adenosylhomocysteine to regulate genes involved in recycling thismetabolite which is produced when S-adenosylmethionine is used inmethylation reactions.

In some embodiments, the riboswitch is a SAM riboswitch, which bindsS-adenosyl methionine (SAM) to regulate methionine and SAM biosynthesisand transport. Three distinct SAM riboswitches are known: SAM-I(originally called S-box), SAM-II and the SMK box riboswitch. SAM-I iswidespread in bacteria, but SAM-II is found only in α-, β- and a fewγ-proteobacteria. The SMK box riboswitch is found only in the orderLactobacillales. These three varieties of riboswitch have no obvioussimilarities in terms of sequence or structure. A fourth variety,SAM-IV, appears to have a similar ligand-binding core to that of SAM-I,but in the context of a distinct scaffold.

In some embodiments, the riboswitch is a SAM-SAH riboswitch, which bindsboth SAM and SAH with similar affinities. Since they are always found ina position to regulate genes encoding methionine adenosyltransferase, itwas proposed that only their binding to SAM is physiologically relevant.

In some embodiments, the riboswitch is a Tetrahydrofolate riboswitch,which binds tetrahydrofolate to regulate synthesis and transport genes.

In some embodiments, the riboswitch is a theophylline binding riboswitchor a thymine pyrophosphate binding riboswitch.

In some embodiments, the riboswitch is a T. tengcongensis glmS catalyticriboswitch, which senses glucosamine-6 phosphate (Klein andFerre-D′Amare 2006).

In some embodiments, the riboswitch is a TPP riboswitch (also THI-box),which binds thiamine pyrophosphate (TPP) to regulate thiaminebiosynthesis and transport, as well as transport of similar metabolites.It is the only riboswitch found so far in eukaryotes.

In some embodiments, the riboswitch is a Moco riboswitch, which bindsmolybdenum cofactor, to regulate genes involved in biosynthesis andtransport of this coenzyme, as well as enzymes that use it or itsderivatives as a cofactor.

In some embodiments, the riboswitch is a Adenine sensing add-Ariboswitch, found in the 5′ UTR of the adenine deaminase encoding geneof Vibrio vulnificus.

Aptazyme

In some embodiments, the circular polyribonucleotide comprises anaptazyme. Aptazyme is a switch for conditional expression in which anaptamer region is used as an allosteric control element and coupled to aregion of catalytic RNA (a “ribozyme” as described below). In someembodiments, the aptazyme is active in cell type specific translation.In some embodiments, the aptazyme is active under cell state specifictranslation, e.g., virally infected cells or in the presence of viralnucleic acids or viral proteins.

A ribozyme (from ribonucleic acid enzyme, also called RNA enzyme orcatalytic RNA) is a RNA molecule that catalyzes a chemical reaction.Many natural ribozymes catalyze either the hydrolysis of one of theirown phosphodiester bonds, or the hydrolysis of bonds in other RNAs, butthey have also been found to catalyze the aminotransferase activity ofthe ribosome. More recently it has been shown that catalytic RNAs can be“evolved” by in vitro methods [1. Agresti J J, Kelly B T, Jaschke A,Griffiths A D: Selection of ribozymes that catalyse multiple-turnoverDiels-Alder cycloadditions by using in vitro compartmentalization. ProcNatl Acad Sci USA 2005, 102:16170-16175; 2. Sooter L J, Riedel T,Davidson E A, Levy M, Cox J C, Ellington A D: Toward automated nucleicacid enzyme selection. Biological Chemistry 2001, 382(9):1327-1334.].Winkler et al. have shown [Winkler W C, Nahvi A, Roth A, Collins J A,Breaker R R: Control of gene expression by a naturalmetabolite-responsive ribozyme. Nature 2004, 428:281-286.] that, similarto riboswitch activity discussed above, ribozymes and their reactionproducts can regulate gene expression. In the context of the instantinvention, it may be particularly advantageous to place a catalytic RNAor ribozyme within a larger non-coding RNA such that the ribozyme ispresent at many copies within the cell for the purposes of chemicaltransformation of a molecule from a bulk volume. Furthermore, encodingboth aptamers and ribozymes in the same non-coding RNA may beparticularly advantageous.

Some nonlimiting examples of ribozymes include hammerhead ribozyme, VLribozyme, leadzyme, hairpin ribozyme.

In some embodiments, the aptazyme is a ribozyme that can cleave RNAsequences and which can be regulated as a result of bindingligand/modulator. The ribozyme may also be a self-cleaving ribozyme. Assuch, they combine the properties of ribozymes and aptamers. Aptazymesoffer advantages over conventional aptamers due to their potential foractivity in trans, the fact that they act catalytically to inactivateexpression and that inactivation, due to cleavage of their own orheterologous transcript, is irreversible.

In some embodiments, the aptazyme is included in an untranslated regionof the circular polyribonucleotide and in the absence ofligand/modulator is inactive, allowing expression of the transgene.Expression can be turned off (or down-regulated) by addition of theligand. It should be noted that aptazymes which are downregulated inresponse to the presence of a particular modulator can be used incontrol systems where upregulation of gene expression in response tomodulator is desired.

Aptazymes may also permit development of systems for self-regulation ofcircular polyribonucleotide expression. For example, the protein productof the circular polyribonucleotide is the rate determining enzyme in thesynthesis of a particular small molecule could be modified to include anaptazyme selected to have increased catalytic activity in the presenceof that molecule, thereby providing an autoregulatory feedback loop forits synthesis. Alternatively, the aptazyme activity can be selected tobe sensitive to accumulation of the protein product from the circularpolyribonucleotide, or any other cellular macromolecule.

In some embodiments, the circular polyribonucleotide may include anaptamer sequence. Some nonlimiting examples include an RNA aptamerbinding lysozyme, a Toggle-25t which is an RNA aptamer that includes2′fluoropyrimidine nucleotides bind thrombins with high specificity andaffinity, RNATat that binds human immunodeficiency virus trans-actingresponsive element (HIV TAR), RNA aptamer-binding hemin, RNAaptamer-binding interferon γ, RNA aptamer binding vascular endothelialgrowth factor (VEGF), RNA aptamer binding prostate specific antigen(PSA), RNA aptamer binding dopamine, and RNA aptamer binding thenon-classical oncogene, heat shock factor 1 (HSF1).

Circularization

In one embodiment, a linear circular polyribonucleotide may be cyclized,or concatemerized. In some embodiments, the linear circularpolyribonucleotide may be cyclized in vitro prior to formulation and/ordelivery. In some embodiments, the linear circular polyribonucleotidemay be cyclized within a cell.

Extracellular Circularization

In some embodiments, the linear circular polyribonucleotide is cyclized,or concatemerized using a chemical method to form a circularpolyribonucleotide. In some chemical methods, the 5′-end and the 3′-endof the nucleic acid (e.g., a linear circular polyribonucleotide)includes chemically reactive groups that, when close together, may forma new covalent linkage between the 5′-end and the 3′-end of themolecule. The 5′-end may contain an NHS-ester reactive group and the3′-end may contain a 3′-amino-terminated nucleotide such that in anorganic solvent the 3′-amino-terminated nucleotide on the 3′-end of alinear RNA molecule will undergo a nucleophilic attack on the5′-NHS-ester moiety forming a new 5′-/3′-amide bond.

In one embodiment, a DNA or RNA ligase may be used to enzymatically linka 5′-phosphorylated nucleic acid molecule (e.g., a linear circularpolyribonucleotide) to the 3′-hydroxyl group of a nucleic acid (e.g., alinear nucleic acid) forming a new phosphorodiester linkage. In anexample reaction, a linear circular polyribonucleotide is incubated at37° C. for 1 hour with 1-10 units of T4 RNA ligase (New England Biolabs,Ipswich, Mass.) according to the manufacturer's protocol. The ligationreaction may occur in the presence of a linear nucleic acid capable ofbase-pairing with both the 5′- and 3′-region in juxtaposition to assistthe enzymatic ligation reaction. In one embodiment, the ligation issplint ligation. For example, a splint ligase, like SplintR® ligase, canbe used for splint ligation. For splint ligation, a single strandedpolynucleotide (splint), like a single stranded RNA, can be designed tohybridize with both termini of a linear polyribonucleotide, so that thetwo termini can be juxtaposed upon hybridization with thesingle-stranded splint. Splint ligase can thus catalyze the ligation ofthe juxtaposed two termini of the linear polyribonucleotide, generatinga circular polyribonucleotide.

In one embodiment, a DNA or RNA ligase may be used in the synthesis ofthe circular polynucleotides. As a non-limiting example, the ligase maybe a circ ligase or circular ligase.

In one embodiment, either the 5′- or 3′-end of the linear circularpolyribonucleotide can encode a ligase ribozyme sequence such thatduring in vitro transcription, the resultant linear circularpolyribonucleotide includes an active ribozyme sequence capable ofligating the 5′-end of the linear circular polyribonucleotide to the3′-end of the linear circular polyribonucleotide. The ligase ribozymemay be derived from the Group I Intron, Hepatitis Delta Virus, Hairpinribozyme or may be selected by SELEX (systematic evolution of ligands byexponential enrichment). The ribozyme ligase reaction may take 1 to 24hours at temperatures between 0 and 37° C.

In one embodiment, a linear circular polyribonucleotide may be cyclizedor concatermerized by using at least one non-nucleic acid moiety. In oneaspect, the at least one non-nucleic acid moiety may react with regionsor features near the 5′ terminus and/or near the 3′ terminus of thelinear circular polyribonucleotide in order to cyclize or concatermerizethe linear circular polyribonucleotide. In another aspect, the at leastone non-nucleic acid moiety may be located in or linked to or near the5′ terminus and/or the 3′ terminus of the linear circularpolyribonucleotide. The non-nucleic acid moieties contemplated may behomologous or heterologous. As a non-limiting example, the non-nucleicacid moiety may be a linkage such as a hydrophobic linkage, ioniclinkage, a biodegradable linkage and/or a cleavable linkage. As anothernon-limiting example, the non-nucleic acid moiety is a ligation moiety.As yet another non-limiting example, the non-nucleic acid moiety may bean oligonucleotide or a peptide moiety, such as an apatamer or anon-nucleic acid linker as described herein.

In one embodiment, a linear circular polyribonucleotide may be cyclizedor concatermerized due to a non-nucleic acid moiety that causes anattraction between atoms, molecular surfaces at, near or linked to the5′ and 3′ ends of the linear circular polyribonucleotide. As anon-limiting example, one or more linear circular polyribonucleotidesmay be cyclized or concatermized by intermolecular forces orintramolecular forces. Non-limiting examples of intermolecular forcesinclude dipole-dipole forces, dipole-induced dipole forces, induceddipole-induced dipole forces, Van der Waals forces, and Londondispersion forces. Non-limiting examples of intramolecular forcesinclude covalent bonds, metallic bonds, ionic bonds, resonant bonds,agnostic bonds, dipolar bonds, conjugation, hyperconjugation andantibonding.

In one embodiment, the linear circular polyribonucleotide may comprise aribozyme RNA sequence near the 5′ terminus and near the 3′ terminus. Theribozyme RNA sequence may covalently link to a peptide when the sequenceis exposed to the remainder of the ribozyme. In one aspect, the peptidescovalently linked to the ribozyme RNA sequence near the 5′ terminus andthe 3′ terminus may associate with each other causing a linear circularpolyribonucleotide to cyclize or concatemerize. In another aspect, thepeptides covalently linked to the ribozyme RNA near the 5′ terminus andthe 3′ terminus may cause the linear primary construct or linear mRNA tocyclize or concatemerize after being subjected to ligated using variousmethods known in the art such as, but not limited to, protein ligation.Non-limiting examples of ribozymes for use in the linear primaryconstructs or linear RNA of the present invention or a non-exhaustivelisting of methods to incorporate and/or covalently link peptides aredescribed in US patent application No. US20030082768, the contents ofwhich is here in incorporated by reference in its entirety.

In some embodiments, the linear circular polyribonucleotide may includea 5′ triphosphate of the nucleic acid converted into a 5′ monophosphate,e.g., by contacting the 5′ triphosphate with RNA 5′ pyrophosphohydrolase(RppH) or an ATP diphosphohydrolase (apyrase). Alternately, convertingthe 5′ triphosphate of the linear circular polyribonucleotide into a 5′monophosphate may occur by a two-step reaction comprising: (a)contacting the 5′ nucleotide of the linear circular polyribonucleotidewith a phosphatase (e.g., Antarctic Phosphatase, Shrimp AlkalinePhosphatase, or Calf Intestinal Phosphatase) to remove all threephosphates; and (b) contacting the 5′ nucleotide after step (a) with akinase (e.g., Polynucleotide Kinase) that adds a single phosphate.

In some embodiments, the circularization efficiency of thecircularization methods provided herein is at least about 10%, at leastabout 15%, at least about 20%, at least about 25%, at least about 30%,at least about 35%, at least about 40%, at least about 45%, at leastabout 50%, at least about 60%, at least about 70%, at least about 80%,at least about 90%, at least about 95%, or 100%. In some embodiments,the circularization efficiency of the circularization methods providedherein is at least about 40%.

Splicing Element

In some embodiment, the circular polyribonucleotide includes at leastone splicing element. In a circular polyribonucleotide as providedherein, a splicing element can be a complete splicing element that canmediate splicing of the circular polyribonucleotide. Alternatively, thespicing element can also be a residual splicing element from a completedsplicing event. For instance, in some cases, a splicing element of alinear polyribonucleotide can mediate a splicing event that results incircularization of the linear polyribonucleotide, thereby the resultantcircular polyribonucleotide comprises a residual splicing element fromsuch splicing-mediated circularization event. In some cases, theresidual splicing element is not able to mediate any splicing. In othercases, the residual splicing element can still mediate splicing undercertain circumstances. In some embodiments, the splicing element isadjacent to at least one expression sequence. In some embodiments, thecircular polyribonucleotide includes a splicing element adjacent eachexpression sequence. In some embodiments, the splicing element is on oneor both sides of each expression sequence, leading to separation of theexpression products, e.g., peptide(s) and or polypeptide(s).

In some embodiments, the circular polyribonucleotide includes aninternal splicing element that when replicated the spliced ends arejoined together. Some examples may include miniature introns (<100 nt)with splice site sequences and short inverted repeats (30-40 nt) such asAluSq2, AluJr, and AluSz, inverted sequences in flanking introns, Aluelements in flanking introns, and motifs found in (suptable4 enrichedmotifs) cis-sequence elements proximal to backsplice events such assequences in the 200 bp preceding (upstream of) or following (downstreamfrom) a backsplice site with flanking exons. In some embodiments, thecircular polyribonucleotide includes at least one repetitive nucleotidesequence described elsewhere herein as an internal splicing element. Insuch embodiments, the repetitive nucleotide sequence may includerepeated sequences from the Alu family of introns. In some embodiments,a splicing-related ribosome binding protein can regulate circularpolyribonucleotide biogenesis (e.g. the Muscleblind and Quaking (QKI)splicing factors).

In some embodiments, the circular polyribonucleotide may includecanonical splice sites that flank head-to-tail junctions of the circularpolyribonucleotide.

In some embodiments, the circular polyribonucleotide may include abulge-helix-bulge motif, comprising a 4-base pair stem flanked by two3-nucleotide bulges. Cleavage occurs at a site in the bulge region,generating characteristic fragments with terminal 5′-hydroxyl group and2′, 3′-cyclic phosphate. Circularization proceeds by nucleophilic attackof the 5′-OH group onto the 2′, 3′-cyclic phosphate of the same moleculeforming a 3′, 5′-phosphodiester bridge.

In some embodiments, the circular polyribonucleotide may include amultimeric repeating RNA sequence that harbors a HPR element. The HPRcomprises a 2′,3′-cyclic phosphate and a 5′-OH termini. The HPR elementself-processes the 5′- and 3′-ends of the linear circularpolyribonucleotide, thereby ligating the ends together.

In some embodiments, the circular polyribonucleotide may include asequence that mediates self-ligation. In one embodiment, the circularpolyribonucleotide may include a HDV sequence (e.g., HDV replicationdomain conserved sequence,

(SEQ ID NO: 102) GGCUCAUCUCGACAAGAGGCGGCAGUCCUCAGUACUCUUACUCUUUUCUGUAAAGAGGAGACUGCUGGACUCGCCGCCCAAGUUCGAGCAUGAGCC or (SEQ ID NO: 103)GGCUAGAGGCGGCAGUCCUCAGUACUCUUACUCUUUUCUGUAAAGAGGAGACUGCUGGACUCGCCGCCCGAGCC)to self-ligate. In one embodiment, the circular polyribonucleotide mayinclude loop E sequence (e.g. in PSTVd) to self-ligate. In anotherembodiment, the circular polyribonucleotide may include aself-circularizing intron, e.g., a 5′ and 3′ slice junction, or aself-circularizing catalytic intron such as a Group I, Group II or GroupIII Introns. Nonlimiting examples of group I intron self-splicingsequences may include self-splicing permuted intron-exon sequencesderived from T4 bacteriophage gene td, and the intervening sequence(IVS) rRNA of Tetrahymena.Other Circularization Methods

In some embodiments, linear circular polyribonucleotides may includecomplementary sequences, including either repetitive or nonrepetitivenucleic acid sequences within individual introns or across flankingintrons. Repetitive nucleic acid sequence are sequences that occurwithin a segment of the circular polyribonucleotide. In someembodiments, the circular polyribonucleotide includes a repetitivenucleic acid sequence. In some embodiments, the repetitive nucleotidesequence includes poly CA or poly UG sequences. In some embodiments, thecircular polyribonucleotide includes at least one repetitive nucleicacid sequence that hybridizes to a complementary repetitive nucleic acidsequence in another segment of the circular polyribonucleotide, with thehybridized segment forming an internal double strand. In someembodiments, repetitive nucleic acid sequences and complementaryrepetitive nucleic acid sequences from two separate circularpolyribonucleotides hybridize to generate a single circularizedpolyribonucleotide, with the hybridized segments forming internal doublestrands. In some embodiments, the complementary sequences are found atthe 5′ and 3′ ends of the linear circular polyribonucleotides. In someembodiments, the complementary sequences include about 3, 4, 5, 6, 7, 8,9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26,27, 28, 29, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100,or more paired nucleotides.

In some embodiments, chemical methods of circularization may be used togenerate the circular polyribonucleotide. Such methods may include, butare not limited to click chemistry (e.g., alkyne and azide basedmethods, or clickable bases), olefin metathesis, phosphoramidateligation, hemiaminal-imine crosslinking, base modification, and anycombination thereof.

In some embodiments, enzymatic methods of circularization may be used togenerate the circular polyribonucleotide. In some embodiments, aligation enzyme, e.g., DNA or RNA ligase, may be used to generate atemplate of the circular polyribonuclease or complement, a complementarystrand of the circular polyribonuclease, or the circularpolyribonuclease.

Circularization of the circular polyribonucleotide may be accomplishedby methods known in the art, for example, those described in “RNAcircularization strategies in vivo and in vitro” by Petkovic and Mullerfrom Nucleic Acids Res, 2015, 43(4): 2454-2465, and “In vitrocircularization of RNA” by Muller and Appel, from RNA Biol, 2017,14(8):1018-1027.

Replication Element

The circular polyribonucleotide may encode a sequence and/or motifsuseful for replication. Replication of a circular polyribonucleotide mayoccur by generating a complement circular polyribonucleotide. In someembodiments, the circular polyribonucleotide includes a motif toinitiate transcription, where transcription is driven by eitherendogenous cellular machinery (DNA-dependent RNA polymerase) or anRNA-depended RNA polymerase encoded by the circular polyribonucleotide.The product of rolling-circle transcriptional event may be cut by aribozyme to generate either complementary or propagated circularpolyribonucleotide at unit length. The ribozymes may be encoded by thecircular polyribonucleotide, its complement, or by an RNA sequence intrans. In some embodiments, the encoded ribozymes may include a sequenceor motif that regulates (inhibits or promotes) activity of the ribozymeto control circular RNA propagation. In some embodiments, unit-lengthsequences may be ligated into a circular form by a cellular RNA ligase.In some embodiments, the circular polyribonucleotide includes areplication element that aids in self amplification. Examples of suchreplication elements include, but are not limited to, HDV replicationdomains described elsewhere herein, RNA promotor of Potato Spindle TuberViroid (see for example Kolonko 2005 Virology), and replicationcompetent circular RNA sense and/or antisense ribozymes such as

antigenomic 5′- CGGGUCGGCAUGGCAUCUCCACCUCCUCGCGGUCCGACCUGGGCAUCCGAAGGAGGACGCACGUCCACUCGGAUGGCUAAGGGAGAGCCA-3′(SEQID NO: 104) or genomic 5′-UGGCCGGCAUGGUCCCAGCCUCCUCGCUGGCGCCGGCUGGGCAACAUUCCGAGGGGACCGUCCCCUCGGUAAUGGCGAAUGGGACCCA-3′ (SEQ ID NO: 105).

In some embodiments, the circular polyribonucleotide includes at leastone stagger element as described herein to aid in replication. A staggerelement within the circular polyribonucleotide can cleave longtranscripts replicated from the circular polyribonucleotide to aspecific length that could subsequently circularize to form a complementto the circular polyribonucleotide.

In another embodiment, the circular polyribonucleotide includes at leastone ribozyme sequence to cleave long transcripts replicated from thecircular polyribonucleotide to a specific length, where another encodedribozyme cuts the transcripts at the ribozyme sequence. Circularizationforms a complement to the circular polyribonucleotide.

In some embodiments, the circular polyribonucleotide is substantiallyresistant to degradation, e.g., by exonucleases.

In some embodiments, the circular polyribonucleotide replicates within acell. In some embodiments, the circular polyribonucleotide replicateswithin in a cell at a rate of between about 10%-20%, 20%-30%, 30%-40%,40%-50%, 50%-60%, 60%-70%, 70%-75%, 75%-80%, 80%-85%, 85%-90%, 90%-95%,95%-99%, or any percentage therebetween. In some embodiments, thecircular polyribonucleotide is replicated within a cell and is passed todaughter cells. In some embodiments, a cell passes at least one circularpolyribonucleotide to daughter cells with an efficiency of at least 25%,50%, 60%, 70%, 80%, 85%, 90%, 95%, or 99%. In some embodiments, cellundergoing meiosis passes the circular polyribonucleotide to daughtercells with an efficiency of at least 25%, 50%, 60%, 70%, 80%, 85%, 90%,95%, or 99%. In some embodiments, a cell undergoing mitosis passes thecircular polyribonucleotide to daughter cells with an efficiency of atleast 25%, 50%, 60%, 70%, 80%, 85%, 90%, 95%, or 99%.

In some embodiments, the circular polyribonucleotide replicates withinthe host cell. In one embodiment, the circular polyribonucleotide iscapable of replicating in a mammalian cell, e.g., human cell.

While in some embodiments the circular polyribonucleotide replicates inthe host cell, the circular polyribonucleotide does not integrate intothe genome of the host, e.g., with the host's chromosomes. In someembodiments, the circular polyribonucleotide has a negligiblerecombination frequency, e.g., with the host's chromosomes. In someembodiments, the circular polyribonucleotide has a recombinationfrequency, e.g., less than about 1.0 cM/Mb, 0.9 cM/Mb, 0.8 cM/Mb, 0.7cM/Mb, 0.6 cM/Mb, 0.5 cM/Mb, 0.4 cM/Mb, 0.3 cM/Mb, 0.2 cM/Mb, 0.1 cM/Mb,or less, e.g., with the host's chromosomes.

Other Sequences

In some embodiments, the circular polyribonucleotide further includesanother nucleic acid sequence. In some embodiments, the circularpolyribonucleotide may comprise other sequences that include DNA, RNA,or artificial nucleic acids. The other sequences may include, but arenot limited to, genomic DNA, cDNA, or sequences that encode tRNA, mRNA,rRNA, miRNA, gRNA, siRNA, or other RNAi molecules. In one embodiment,the circular polyribonucleotide includes an siRNA to target a differentloci of the same gene expression product as the circularpolyribonucleotide. In one embodiment, the circular polyribonucleotideincludes an siRNA to target a different gene expression product as thecircular polyribonucleotide.

In some embodiments, the circular polyribonucleotide lacks a 5′-UTR. Insome embodiments, the circular polyribonucleotide lacks a 3′-UTR. Insome embodiments, the circular polyribonucleotide lacks a poly-Asequence. In some embodiments, the circular polyribonucleotide lacks atermination element. In some embodiments, the circularpolyribonucleotide lacks an internal ribosomal entry site. In someembodiments, the circular polyribonucleotide lacks degradationsusceptibility by exonucleases. In some embodiments, the fact that thecircular polyribonucleotide lacks degradation susceptibility can meanthat the circular polyribonucleotide is not degraded by an exonuclease,or only degraded in the presence of an exonuclease to a limited extentthat is comparable to or similar to in the absence of exonuclease. Insome embodiments, the circular polyribonucleotide lacks degradation byexonucleases. In some embodiments, the circular polyribonucleotide hasreduced degradation when exposed to exonuclease. In some embodiments,the circular polyribonucleotide lacks binding to a cap-binding proteinIn some embodiments, the circular polyribonucleotide lacks a 5′ cap.

In some embodiments, the circular polyribonucleotide lacks a 5′-UTR andis competent for protein express from its one or more expressionsequences. In some embodiments, the circular polyribonucleotide lacks a3′-UTR and is competent for protein express from its one or moreexpression sequences. In some embodiments, the circularpolyribonucleotide lacks a poly-A sequence and is competent for proteinexpress from its one or more expression sequences. In some embodiments,the circular polyribonucleotide lacks a termination element and iscompetent for protein express from its one or more expression sequences.In some embodiments, the circular polyribonucleotide lacks an internalribosomal entry site and is competent for protein express from its oneor more expression sequences. In some embodiments, the circularpolyribonucleotide lacks a cap and is competent for protein express fromits one or more expression sequences. In some embodiments, the circularpolyribonucleotide lacks a 5′-UTR, a 3′-UTR, and an IRES, and iscompetent for protein express from its one or more expression sequences.In some embodiments, the circular polyribonucleotide comprises one ormore of the following sequences: a sequence that encodes one or moremiRNAs, a sequence that encodes one or more replication proteins, asequence that encodes an exogenous gene, a sequence that encodes atherapeutic, a regulatory element (e.g., translation modulator, e.g.,translation enhancer or suppressor), a translation initiation sequence,one or more regulatory nucleic acids that targets endogenous genes(siRNA, lncRNAs, shRNA), and a sequence that encodes a therapeutic mRNAor protein.

The other sequence may have a length from about 2 to about 10000 nts,about 2 to about 5000 nts, about 10 to about 100 nts, about 50 to about150 nts, about 100 to about 200 nts, about 150 to about 250 nts, about200 to about 300 nts, about 250 to about 350 nts, about 300 to about 500nts, about 10 to about 1000 nts, about 50 to about 1000 nts, about 100to about 1000 nts, about 1000 to about 2000 nts, about 2000 to about3000 nts, about 3000 to about 4000 nts, about 4000 to about 5000 nts, orany range therebetween.

As a result of its circularization, the circular polyribonucleotide mayinclude certain characteristics that distinguish it from linear RNA. Forexample, the circular polyribonucleotide is less susceptible todegradation by exonuclease as compared to linear RNA. As such, thecircular polyribonucleotide is more stable than a linear RNA, especiallywhen incubated in the presence of an exonuclease. The increasedstability of the circular polyribonucleotide compared with linear RNAmakes circular polyribonucleotide more useful as a cell transformingreagent to produce polypeptides and can be stored more easily and forlonger than linear RNA. The stability of the circular polyribonucleotidetreated with exonuclease can be tested using methods standard in artwhich determine whether RNA degradation has occurred (e.g., by gelelectrophoresis).

Moreover, unlike linear RNA, the circular polyribonucleotide is lesssusceptible to dephosphorylation when the circular polyribonucleotide isincubated with phosphatase, such as calf intestine phosphatase.Nucleotide spacer sequences

In some embodiments, the circular polyribonucleotide comprises a spacersequence.

In some embodiments, the circular polyribonucleotide comprises at leastone spacer sequence. In some embodiments, the circularpolyribonucleotide comprises 1, 2, 3, 4, 5, 6, 7 or more spacersequences.

In some embodiments, the circular polyribonucleotide comprises a ratioof spacer sequence to non-spacer sequence of the circularpolyribonucleotide, e.g., expression sequences, of about 0.05:1, about0.06:1, about 0.07:1,about 0.08:1, about 0.09:1, about 0.1:1, about0.12:1, about 0.125:1, about 0.15:1, about 0.175:1, about 0.2:1, about0.225:1, about 0.25:1, about 0.3:1, about 0.35:1, about 0.4:1, about0.45:1, about 0.5:1, about 0.55:1, about 0.6:1, about 0.65:1, about0.7:1, about 0.75:1, about 0.8:1, about 0.85:1, about 0.9:1, about0.95:1, about 0.98:1, about 1:1, about 1.02:1, about 1.05:1, about1.1:1, about 1.15:1, about 1.2:1, about 1.25:1, about 1.3:1, about1.35:1, about 1.4:1, about 1.45:1, about 1.5:1, about 1.55:1, about1.6:1, about 1.65:1, about 1.7:1, about 1.75:1, about 1.8:1, about1.85:1, about 1.9:1, about 1.95:1, about 1.975:1, about 1.98:1, or about2:1.

In some embodiments, the spacer sequence comprises a ratio of spacersequence to a downstream (e.g., 3′ of the spacer sequence) non-spacerelement of the circular polyribonucleotide of about 0.5:1, about 0.06:1,about 0.07:1,about 0.08:1, about 0.09:1, about 0.1:1, about 0.12:1,about 0.125:1, about 0.15:1, about 0.175:1, about 0.2:1, about 0.225:1,about 0.25:1, about 0.3:1, about 0.35:1, about 0.4:1, about 0.45:1,about 0.5:1, about 0.55:1, about 0.6:1, about 0.65:1, about 0.7:1, about0.75:1, about 0.8:1, about 0.85:1, about 0.9:1, about 0.95:1, about0.98:1, about 1:1, about 1.02:1, about 1.05:1, about 1.1:1, about1.15:1, about 1.2:1, about 1.3:1, about 1.4:1, about 1.5:1, about 1.6:1,about 1.7:1, about 1.8:1, about 1.9:1, about 1.95:1, about 1.975:1,about 1.98:1, about 2.1:1, about 2.2:1, about 2.3:1, about 2.4:1, about2.5:1, about 2.6:1, about 2.7:1, about 2.8:1, about 2.9:1, about 3:1,about 3.1:1, about 3.2:1, about 3.3:1, about 3.4:1, about 3.5:1, about3.6:1, about 3.7:1, about 3.8:1, about 3.85:1,about 3.9:1, about 3.95:1,about 3.98:1, or about 4:1. In some embodiments, the spacer sequencecomprises a ratio of spacer sequence to an upstream (e.g., 5′ of thespacer sequence) non-spacer element of the circular polyribonucleotideof about 0.5:1, about 0.06:1, about 0.07:1,about 0.08:1, about 0.09:1,about 0.1:1, about 0.12:1, about 0.125:1, about 0.15:1, about 0.175:1,about 0.2:1, about 0.225:1, about 0.25:1, about 0.3:1, about 0.35:1,about 0.4:1, about 0.45:1, about 0.5:1, about 0.55:1, about 0.6:1, about0.65:1, about 0.7:1, about 0.75:1, about 0.8:1, about 0.85:1, about0.9:1, about 0.95:1, about 0.98:1, about 1:1, about 1.02:1, about1.05:1, about 1.1:1, about 1.15:1, about 1.2:1, about 1.3:1, about1.4:1, about 1.5:1, about 1.6:1, about 1.7:1, about 1.8:1, about 1.9:1,about 1.95:1, about 1.975:1, about 1.98:1, about 2.1:1, about 2.2:1,about 2.3:1, about 2.4:1, about 2.5:1, about 2.6:1, about 2.7:1, about2.8:1, about 2.9:1, about 3:1, about 3.1:1, about 3.2:1, about 3.3:1,about 3.4:1, about 3.5:1, about 3.6:1, about 3.7:1, about 3.8:1, about3.85:1,about 3.9:1, about 3.95:1, about 3.98:1, or about 4:1.

In some embodiments, the spacer sequence comprises a sequence of atleast 3 ribonucleotides, at least 4 ribonucleotides, at least 5ribonucleotides, at least about 8 ribonucleotides, at least about 10ribonucleotides, at least about 12 ribonucleotides, at least about 15ribonucleotides, at least about 20 ribonucleotides, at least about 25ribonucleotides, at least about 30 ribonucleotides, at least about 40ribonucleotides, at least about 50 ribonucleotides, at least about 60ribonucleotides, at least about 70 ribonucleotides, at least about 80ribonucleotides, at least about 90 ribonucleotides, at least about 100ribonucleotides, at least about 120 ribonucleotides, at least about 150ribonucleotides, at least about 200 ribonucleotides, at least about 250ribonucleotides, at least about 300 ribonucleotides, at least about 400ribonucleotides, at least about 500 ribonucleotides, at least about 600ribonucleotides, at least about 700 ribonucleotides, at least about 800ribonucleotides, at least about 900 ribonucleotides, or at least about100 ribonucleotides.

In some embodiments, the spacer sequence may be a nucleic acid sequenceor molecule having low GC content, for example less than 65%, 60%, 55%,50%, 55%, 50%, 45%, 40%, 39%, 38%, 37%, 36%, 35%, 34%, 33%, 32%, 31%,30%, 29%, 28%, 27%, 26%, 25%, 24%, 23%, 22%, 20%, 19%, 18%, 17%, 16%,15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2% or 1%,across the full length of the spacer, or across at least 50%, 60%, 70%,80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% contiguousnucleic acid residues of the spacer. In some embodiments, the spacersequence may comprise at least 95%, 90%, 85%, 80%, 75%, 70%, 65%, 60%,55%, 50%, 55%, 50%, 45%, 40%, 35%, 30%, 20% or any percentagetherebetween of adenine ribonucleotides. In some embodiments, the spacersequence comprises at least 5 or more adenine ribonucleotides in a row.In some embodiments, the spacer sequence comprises at least 6 adenineribonucleotides in a row, at least 7 adenine ribonucleotides in a row,at least 8 ribonucleotides, at least about 10 adenine ribonucleotides ina row, at least about 12 adenine ribonucleotides in a row, at leastabout 15 adenine ribonucleotides in a row, at least about 20 adenineribonucleotides in a row, at least about 25 adenine ribonucleotides in arow, at least about 30 adenine ribonucleotides in a row, at least about40 adenine ribonucleotides in a row, at least about 50 adenineribonucleotides in a row, at least about 60 adenine ribonucleotides in arow, at least about 70 adenine ribonucleotides in a row, at least about80 adenine ribonucleotides in a row, at least about 90 adenineribonucleotides in a row, at least about 95 adenine ribonucleotides in arow, at least about 100 adenine ribonucleotides in a row, at least about150 adenine ribonucleotides in a row, at least about 200 adenineribonucleotides in a row, at least about 250 adenine ribonucleotides ina row, at least about 300 adenine ribonucleotides in a row, at leastabout 350 adenine ribonucleotides in a row, at least about 400 adenineribonucleotides in a row, at least about 450 adenine ribonucleotides ina row, at least about 500 adenine ribonucleotides in a row, at leastabout 550 adenine ribonucleotides in a row, at least about 600 adenineribonucleotides in a row, at least about 700 adenine ribonucleotides ina row, at least about 800 adenine ribonucleotides in a row, at leastabout 900 adenine ribonucleotides in a row, or at least about 1000adenine ribonucleotides in a row.

In some embodiments, the spacer sequence is situated between one or moreelements. In some embodiments, the spacer sequence providesconformational flexibility between the elements. In some embodiments,the conformational flexibility is due to the spacer sequence beingsubstantially free of a secondary structure. In some embodiments, thespacer sequence is substantially free of a secondary structure, such asless than 40 kcal/mol, less than −39, −38, −37, −36, −35, −34, −33, −32,−31, −30, −29, −28, −27, −26, −25, −24, −23, −22, −20, −19, −18, −17,−16, −15, −14, −13, −12, −11, −10, −9, −8, −7, −6, −5, −4, −3, −2 or −1kcal/mol. The spacer may include a nucleic acid, such as DNA or RNA.

In some embodiments, the spacer sequence may encode an RNA sequence, andpreferably a protein or peptide sequence, including a secretion signalpeptide.

In some embodiments, the spacer sequence may be non-coding. Where thespacer is a non-coding sequence, a translation initiation sequence maybe provided in the coding sequence of an adjacent sequence. In someembodiments, it is envisaged that the first nucleic acid residue of thecoding sequence may be the A residue of a translation initiationsequence, such as AUG. Where the spacer encodes an RNA or protein orpeptide sequence, a translation initiation sequence may be provided inthe spacer sequence.

In some embodiments, the spacer is operably linked to another sequencedescribed herein.

Non-Nucleic Acid Linkers

The circular polyribonucleotide described herein may also comprise anon-nucleic acid linker. In some embodiments, the circularpolyribonucleotide described herein has a non-nucleic acid linkerbetween one or more of the sequences or elements described herein. Inone embodiment, one or more sequences or elements described herein arelinked with the linker. The non-nucleic acid linker may be a chemicalbond, e.g., one or more covalent bonds or non-covalent bonds. In someembodiments, the non-nucleic acid linker is a peptide or protein linker.Such a linker may be between 2-30 amino acids, or longer. The linkerincludes flexible, rigid or cleavable linkers described herein.

The most commonly used flexible linkers have sequences consistingprimarily of stretches of Gly and Ser residues (“GS” linker). Flexiblelinkers may be useful for joining domains that require a certain degreeof movement or interaction and may include small, non-polar (e.g. Gly)or polar (e.g. Ser or Thr) amino acids. Incorporation of Ser or Thr canalso maintain the stability of the linker in aqueous solutions byforming hydrogen bonds with the water molecules, and therefore reduceunfavorable interactions between the linker and the protein moieties.

Rigid linkers are useful to keep a fixed distance between domains and tomaintain their independent functions. Rigid linkers may also be usefulwhen a spatial separation of the domains is critical to preserve thestability or bioactivity of one or more components in the fusion. Rigidlinkers may have an alpha helix-structure or Pro-rich sequence,(XP)_(n), with X designating any amino acid, preferably Ala, Lys, orGlu.

Cleavable linkers may release free functional domains in vivo. In someembodiments, linkers may be cleaved under specific conditions, such asthe presence of reducing reagents or proteases. In vivo cleavablelinkers may utilize the reversible nature of a disulfide bond. Oneexample includes a thrombin-sensitive sequence (e.g., PRS) between thetwo Cys residues. In vitro thrombin treatment of CPRSC (SEQ ID NO: 131)results in the cleavage of the thrombin-sensitive sequence, while thereversible disulfide linkage remains intact. Such linkers are known anddescribed, e.g., in Chen et al. 2013. Fusion Protein Linkers: Property,Design and Functionality. Adv Drug Deliv Rev. 65(10): 1357-1369. In vivocleavage of linkers in fusions may also be carried out by proteases thatare expressed in vivo under pathological conditions (e.g. cancer orinflammation), in specific cells or tissues, or constrained withincertain cellular compartments. The specificity of many proteases offersslower cleavage of the linker in constrained compartments.

Examples of linking molecules include a hydrophobic linker, such as anegatively charged sulfonate group; lipids, such as a poly (—CH₂—)hydrocarbon chains, such as polyethylene glycol (PEG) group, unsaturatedvariants thereof, hydroxylated variants thereof, amidated or otherwiseN-containing variants thereof, noncarbon linkers; carbohydrate linkers;phosphodiester linkers, or other molecule capable of covalently linkingtwo or more polypeptides. Non-covalent linkers are also included, suchas hydrophobic lipid globules to which the polypeptide is linked, forexample through a hydrophobic region of the polypeptide or a hydrophobicextension of the polypeptide, such as a series of residues rich inleucine, isoleucine, valine, or perhaps also alanine, phenylalanine, oreven tyrosine, methionine, glycine or other hydrophobic residue. Thepolypeptide may be linked using charge-based chemistry, such that apositively charged moiety of the polypeptide is linked to a negativecharge of another polypeptide or nucleic acid.

Stability/Half-Life

In some embodiments, the circular polyribonucleotide provided herein hasincrease half-life over a reference, e.g., a linear polyribonucleotidehaving the same nucleotide sequence but is not circularized (linearcounterpart). In some embodiments, the circular polyribonucleotide issubstantially resistant to degradation, e.g., exonuclease. In someembodiments, the circular polyribonucleotide is resistant toself-degradation. In some embodiments, the circular polyribonucleotidelacks an enzymatic cleavage site, e.g., a dicer cleavage site. In someembodiments, the circular polyribonucleotide has a half-life at leastabout 5%, at least about 10%, at least about 20%, at least about 30%, atleast about 40%, at least about 50%, at least about 60%, at least about70%, at least about 80%, at least about 90%, at least about 100%, atleast about 120%, at least about 140%, at least about 150%, at leastabout 160%, at least about 180%, at least about 200%, at least about300%, at least about 400%, at least about 500%, at least about 600%, atleast about 700% at least about 800%, at least about 900%, at leastabout 1000% or at least about 10000%, longer than a reference, e.g., alinear counterpart.

In some embodiments, the circular polyribonucleotide persists in a cellduring cell division. In some embodiments, the circularpolyribonucleotide persists in daughter cells after mitosis. In someembodiments, the circular polyribonucleotide is replicated within a celland is passed to daughter cells. In some embodiments, the circularpolyribonucleotide comprises a replication element that mediatesself-replication of the circular polyribonucleotide. In someembodiments, the replication element mediates transcription of thecircular polyribonucleotide into a linear polyribonucleotide that iscomplementary to the circular polyribonucleotide (linear complementary).In some embodiments, the linear complementary polyribonucleotide can becircularized in vivo in cells into a complementary circularpolyribonucleotide. In some embodiments, the complementarypolyribonucleotide can further self-replicate into another circularpolyribonucleotide, which has the same or similar nucleotide sequence asthe starting circular polyribonucleotide. One exemplary self-replicationelement includes HDV replication domain (as described by Beeharry et al,Virol, 2014, 450-451:165-173). In some embodiments, a cell passes atleast one circular polyribonucleotide to daughter cells with anefficiency of at least 25%, 50%, 60%, 70%, 80%, 85%, 90%, 95%, or 99%.In some embodiments, cell undergoing meiosis passes the circularpolyribonucleotide to daughter cells with an efficiency of at least 25%,50%, 60%, 70%, 80%, 85%, 90%, 95%, or 99%. In some embodiments, a cellundergoing mitosis passes the circular polyribonucleotide to daughtercells with an efficiency of at least 25%, 50%, 60%, 70%, 80%, 85%, 90%,95%, or 99%.

Modifications

The circular polyribonucleotide may include one or more substitutions,insertions and/or additions, deletions, and covalent modifications withrespect to reference sequences, in particular, the parentpolyribonucleotide, are included within the scope of this invention.

In some embodiments, the circular polyribonucleotide includes one ormore post-transcriptional modifications (e.g., capping, cleavage,polyadenylation, splicing, poly-A sequence, methylation, acylation,phosphorylation, methylation of lysine and arginine residues,acetylation, and nitrosylation of thiol groups and tyrosine residues,etc). The one or more post-transcriptional modifications can be anypost-transcriptional modification, such as any of the more than onehundred different nucleoside modifications that have been identified inRNA (Rozenski, J, Crain, P, and McCloskey, J. (1999). The RNAModification Database: 1999 update. Nucl Acids Res 27: 196-197) In someembodiments, the first isolated nucleic acid comprises messenger RNA(mRNA). In some embodiments, the mRNA comprises at least one nucleosideselected from the group consisting of pyridin-4-one ribonucleoside,5-aza-uridine, 2-thio-5-aza-uridine, 2-thiouridine,4-thio-pseudouridine, 2-thio-pseudouridine, 5-hydroxyuridine,3-methyluridine, 5-carboxymethyl-uridine, 1-carboxymethyl-pseudouridine,5-propynyl-uridine, 1-propynyl-pseudouridine, 5-taurinomethyluridine,1-taurinomethyl-pseudouridine, 5-taurinomethyl-2-thio-uridine,1-taurinomethyl-4-thio-uridine, 5-methyl-uridine,1-methyl-pseudouridine, 4-thio-1-methyl-pseudouridine,2-thio-1-methyl-pseudouridine, 1-methyl-1-deaza-pseudouridine,2-thio-1-methyl-1-deaza-pseudouridine, dihydrouridine,dihydropseudouridine, 2-thio-dihydrouridine,2-thio-dihydropseudouridine, 2-methoxyuridine, 2-methoxy-4-thio-uridine,4-methoxy-pseudouridine, and 4-methoxy-2-thio-pseudouridine. In someembodiments, the mRNA comprises at least one nucleoside selected fromthe group consisting of 5-aza-cytidine, pseudoisocytidine,3-methyl-cytidine, N4-acetylcytidine, 5-formylcytidine,N4-methylcytidine, 5-hydroxymethylcytidine, 1-methyl-pseudoisocytidine,pyrrolo-cytidine, pyrrolo-pseudoisocytidine, 2-thio-cytidine,2-thio-5-methyl-cytidine, 4-thio-pseudoisocytidine,4-thio-1-methyl-pseudoisocytidine,4-thio-1-methyl-1-deaza-pseudoisocytidine,1-methyl-1-deaza-pseudoisocytidine, zebularine, 5-aza-zebularine,5-methyl-zebularine, 5-aza-2-thio-zebularine, 2-thio-zebularine,2-methoxy-cytidine, 2-methoxy-5-methyl-cytidine,4-methoxy-pseudoisocytidine, and 4-methoxy-1-methyl-pseudoisocytidine.In some embodiments, the mRNA comprises at least one nucleoside selectedfrom the group consisting of 2-aminopurine, 2, 6-diaminopurine,7-deaza-adenine, 7-deaza-8-aza-adenine, 7-deaza-2-aminopurine,7-deaza-8-aza-2-aminopurine, 7-deaza-2,6-diaminopurine,7-deaza-8-aza-2,6-diaminopurine, 1-methyladenosine, N6-methyladenosine,N6-isopentenyladenosine, N6-(cis-hydroxyisopentenyl)adenosine,2-methylthio-N6-(cis-hydroxyisopentenyl) adenosine,N6-glycinylcarbamoyladenosine, N6-threonylcarbamoyladenosine,2-methylthio-N6-threonyl carbamoyladenosine, N6,N6-dimethyladenosine,7-methyladenine, 2-methylthio-adenine, and 2-methoxy-adenine. In someembodiments, mRNA comprises at least one nucleoside selected from thegroup consisting of inosine, 1-methyl-inosine, wyosine, wybutosine,7-deaza-guanosine, 7-deaza-8-aza-guanosine, 6-thio-guanosine,6-thio-7-deaza-guanosine, 6-thio-7-deaza-8-aza-guanosine,7-methyl-guanosine, 6-thio-7-methyl-guanosine, 7-methylinosine,6-methoxy-guanosine, 1-methylguanosine, N2-methylguanosine,N2,N2-dimethylguanosine, 8-oxo-guanosine, 7-methyl-8-oxo-guanosine,1-methyl-6-thio-guanosine, N2-methyl-6-thio-guanosine, andN2,N2-dimethyl-6-thio-guanosine.

The circular polyribonucleotide may include any useful modification,such as to the sugar, the nucleobase, or the internucleoside linkage(e.g. to a linking phosphate/to a phosphodiester linkage/to thephosphodiester backbone). One or more atoms of a pyrimidine nucleobasemay be replaced or substituted with optionally substituted amino,optionally substituted thiol, optionally substituted alkyl (e.g., methylor ethyl), or halo (e.g., chloro or fluoro). In certain embodiments,modifications (e.g., one or more modifications) are present in each ofthe sugar and the internucleoside linkage. Modifications may bemodifications of ribonucleic acids (RNAs) to deoxyribonucleic acids(DNAs), threose nucleic acids (TNAs), glycol nucleic acids (GNAs),peptide nucleic acids (PNAs), locked nucleic acids (LNAs) or hybridsthereof). Additional modifications are described herein.

In some embodiments, the circular polyribonucleotide includes at leastone N(6)methyladenosine (m6A) modification to increase translationefficiency. In some embodiments, the N(6)methyladenosine (m6A)modification can reduce immunogeneicity of the circularpolyribonucleotide.

In some embodiments, the modification may include a chemical or cellularinduced modification. For example, some nonlimiting examples ofintracellular RNA modifications are described by Lewis and Pan in “RNAmodifications and structures cooperate to guide RNA-proteininteractions” from Nat Reviews Mol Cell Biol, 2017, 18:202-210.

In some embodiments, chemical modifications to the ribonucleotides ofthe circular polyribonucleotide may enhance immune evasion. The circularpolyribonucleotide may be synthesized and/or modified by methods wellestablished in the art, such as those described in “Current protocols innucleic acid chemistry,” Beaucage, S. L. et al. (Eds.), John Wiley &Sons, Inc., New York, N.Y., USA, which is hereby incorporated herein byreference. Modifications include, for example, end modifications, e.g.,5′ end modifications (phosphorylation (mono-, di- and tri-),conjugation, inverted linkages, etc.), 3′ end modifications(conjugation, DNA nucleotides, inverted linkages, etc.), basemodifications (e.g., replacement with stabilizing bases, destabilizingbases, or bases that base pair with an expanded repertoire of partners),removal of bases (abasic nucleotides), or conjugated bases. The modifiedribonucleotide bases may also include 5-methylcytidine andpseudouridine. In some embodiments, base modifications may modulateexpression, immune response, stability, subcellular localization, toname a few functional effects, of the circular polyribonucleotide. Insome embodiments, the modification includes a bi-orthogonal nucleotides,e.g., an unnatural base. See for example, Kimoto et al, Chem Commun(Camb), 2017, 53:12309, DOI: 10.1039/c7cc06661a, which is herebyincorporated by reference.

In some embodiments, sugar modifications (e.g., at the 2′ position or 4′position) or replacement of the sugar one or more ribonucleotides of thecircular polyribonucleotide may, as well as backbone modifications,include modification or replacement of the phosphodiester linkages.Specific examples of circular polyribonucleotide include, but are notlimited to circular polyribonucleotide including modified backbones orno natural internucleoside linkages such as internucleosidemodifications, including modification or replacement of thephosphodiester linkages. Circular polyribonucleotides having modifiedbackbones include, among others, those that do not have a phosphorusatom in the backbone. For the purposes of this application, and assometimes referenced in the art, modified RNAs that do not have aphosphorus atom in their internucleoside backbone can also be consideredto be oligonucleosides. In particular embodiments, the circularpolyribonucleotide will include ribonucleotides with a phosphorus atomin its internucleoside backbone.

Modified circular polyribonucleotide backbones may include, for example,phosphorothioates, chiral phosphorothioates, phosphorodithioates,phosphotriesters, aminoalkylphosphotriesters, methyl and other alkylphosphonates such as 3′-alkylene phosphonates and chiral phosphonates,phosphinates, phosphoramidates such as 3′-amino phosphoramidate andaminoalkylphosphoramidates, thionophosphoramidates,thionoalkylphosphonates, thionoalkylphosphotriesters, andboranophosphates having normal 3′-5′ linkages, 2′-5′ linked analogs ofthese, and those having inverted polarity wherein the adjacent pairs ofnucleoside units are linked 3′-5′ to 5′-3′ or 2′-5′ to 5′-2′. Varioussalts, mixed salts and free acid forms are also included. In someembodiments, the circular polyribonucleotide may be negatively orpositively charged.

The modified nucleotides, which may be incorporated into the circularpolyribonucleotide, can be modified on the internucleoside linkage(e.g., phosphate backbone). Herein, in the context of the polynucleotidebackbone, the phrases “phosphate” and “phosphodiester” are usedinterchangeably. Backbone phosphate groups can be modified by replacingone or more of the oxygen atoms with a different substituent. Further,the modified nucleosides and nucleotides can include the wholesalereplacement of an unmodified phosphate moiety with anotherinternucleoside linkage as described herein. Examples of modifiedphosphate groups include, but are not limited to, phosphorothioate,phosphoroselenates, boranophosphates, boranophosphate esters, hydrogenphosphonates, phosphoramidates, phosphorodiamidates, alkyl or arylphosphonates, and phosphotriesters. Phosphorodithioates have bothnon-linking oxygens replaced by sulfur. The phosphate linker can also bemodified by the replacement of a linking oxygen with nitrogen (bridgedphosphoramidates), sulfur (bridged phosphorothioates), and carbon(bridged methylene-phosphonates).

The a-thio substituted phosphate moiety is provided to confer stabilityto RNA and DNA polymers through the unnatural phosphorothioate backbonelinkages. Phosphorothioate DNA and RNA have increased nucleaseresistance and subsequently a longer half-life in a cellularenvironment. Phosphorothioate linked to the circular polyribonucleotideis expected to reduce the innate immune response through weakerbinding/activation of cellular innate immune molecules.

In specific embodiments, a modified nucleoside includes analpha-thio-nucleoside (e.g., 5′-0-(1-thiophosphate)-adenosine,5′-0-(1-thiophosphate)-cytidine (a-thio-cytidine),5′-0-(1-thiophosphate)-guanosine, 5′-0-(1-thiophosphate)-uridine, or5′-0-(1-thiophosphate)-pseudouridine).

Other internucleoside linkages that may be employed according to thepresent invention, including internucleoside linkages which do notcontain a phosphorous atom, are described herein.

In some embodiments, the circular polyribonucleotide may include one ormore cytotoxic nucleosides. For example, cytotoxic nucleosides may beincorporated into circular polyribonucleotide, such as bifunctionalmodification. Cytotoxic nucleoside may include, but are not limited to,adenosine arabinoside, 5-azacytidine, 4′-thio-aracytidine,cyclopentenylcytosine, cladribine, clofarabine, cytarabine, cytosinearabinoside,1-(2-C-cyano-2-deoxy-beta-D-arabino-pentofuranosyl)-cytosine,decitabine, 5-fluorouracil, fludarabine, floxuridine, gemcitabine, acombination of tegafur and uracil, tegafur((RS)-5-fluoro-l-(tetrahydrofuran-2-yl)pyrimidine-2,4(1H,3H)-dione),troxacitabine, tezacitabine, 2′-deoxy-2′-methylidenecytidine (DMDC), and6-mercaptopurine. Additional examples include fludarabine phosphate,N4-behenoyl-1-beta-D-arabinofuranosylcytosine,N4-octadecyl-1-beta-D-arabinofuranosylcytosine,N4-palmitoyl-1-(2-C-cyano-2-deoxy-beta-D-arabino-pentofuranosyl)cytosine, and P-4055 (cytarabine 5′-elaidic acid ester).

The circular polyribonucleotide may or may not be uniformly modifiedalong the entire length of the molecule. For example, one or more or alltypes of nucleotide (e.g., naturally-occurring nucleotides, purine orpyrimidine, or any one or more or all of A, G, U, C, I, pU) may or maynot be uniformly modified in the circular polyribonucleotide, or in agiven predetermined sequence region thereof. In some embodiments, thecircular polyribonucleotide includes a pseudouridine. In someembodiments, the circular polyribonucleotide includes an inosine, whichmay aid in the immune system characterizing the circularpolyribonucleotide as endogenous versus viral RNAs. The incorporation ofinosine may also mediate improved RNA stability/reduced degradation. Seefor example, Yu, Z. et al. (2015) RNA editing by ADAR1 marks dsRNA as“self”. Cell Res. 25, 1283-1284, which is incorporated by reference inits entirety.

In some embodiments, all nucleotides in the circular polyribonucleotide(or in a given sequence region thereof) are modified. In someembodiments, the modification may include an m6A, which may augmentexpression; an inosine, which may attenuate an immune response;pseudouridine, which may increase RNA stability, or translationalreadthrough (stagger element), an m5C, which may increase stability; anda 2,2,7-trimethylguanosine, which aids subcellular translocation (e.g.,nuclear localization).

Different sugar modifications, nucleotide modifications, and/orinternucleoside linkages (e.g., backbone structures) may exist atvarious positions in the circular polyribonucleotide. One of ordinaryskill in the art will appreciate that the nucleotide analogs or othermodification(s) may be located at any position(s) of the circularpolyribonucleotide, such that the function of the circularpolyribonucleotide is not substantially decreased. A modification mayalso be a non-coding region modification. The circularpolyribonucleotide may include from about 1% to about 100% modifiednucleotides (either in relation to overall nucleotide content, or inrelation to one or more types of nucleotide, i.e. any one or more of A,G, U or C) or any intervening percentage (e.g., from 1% to 20%>, from 1%to 25%, from 1% to 50%, from 1% to 60%, from 1% to 70%, from 1% to 80%,from 1% to 90%, from 1% to 95%, from 10% to 20%, from 10% to 25%, from10% to 50%, from 10% to 60%, from 10% to 70%, from 10% to 80%, from 10%to 90%, from 10% to 95%, from 10% to 100%, from 20% to 25%, from 20% to50%, from 20% to 60%, from 20% to 70%, from 20% to 80%, from 20% to 90%,from 20% to 95%, from 20% to 100%, from 50% to 60%, from 50% to 70%,from 50% to 80%, from 50% to 90%, from 50% to 95%, from 50% to 100%,from 70% to 80%, from 70% to 90%, from 70% to 95%, from 70% to 100%,from 80% to 90%, from 80% to 95%, from 80% to 100%, from 90% to 95%,from 90% to 100%, and from 95% to 100%).

Structure

In some embodiments, the circular polyribonucleotide comprises a higherorder structure, e.g., a secondary or tertiary structure. In someembodiments, complementary segments of the circular polyribonucleotidefold itself into a double stranded segment, held together with hydrogenbonds between pairs, e.g., A-U and C-G. In some embodiments, helices,also known as stems, are formed intra-molecularly, having adouble-stranded segment connected to an end loop. In some embodiments,the circular polyribonucleotide has at least one segment with aquasi-double-stranded secondary structure. In some embodiments, asegment having a quasi-double-stranded secondary structure has at least3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22,23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80,85, 90, 95, 100, or more paired nucleotides. In some embodiments, thecircular polyribonucleotide has one or more segments (e.g., 2, 3, 4, 5,6, or more) having a quasi-double-stranded secondary structure. In someembodiments, the segments are separated by 3, 4, 5, 6, 7, 8, 9, 10, 11,12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29,30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, or morenucleotides.

In some embodiments, one or more sequences of the circularpolyribonucleotide include substantially single stranded vs doublestranded regions. In some embodiments, the ratio of single stranded todouble stranded may influence the functionality of the circularpolyribonucleotide.

In some embodiments, one or more sequences of the circularpolyribonucleotide that are substantially single stranded. In someembodiments, one or more sequences of the circular polyribonucleotidethat are substantially single stranded may include a protein- orRNA-binding site. In some embodiments, the circular polyribonucleotidesequences that are substantially single stranded may be conformationallyflexible to allow for increased interactions. In some embodiments, thesequence of the circular polyribonucleotide is purposefully engineeredto include such secondary structures to bind or increase protein ornucleic acid binding.

In some embodiments, the circular polyribonucleotide sequences that aresubstantially double stranded. In some embodiments, one or moresequences of the circular polyribonucleotide that are substantiallydouble stranded may include a conformational recognition site, e.g., ariboswitch or aptazyme. In some embodiments, the circularpolyribonucleotide sequences that are substantially double stranded maybe conformationally rigid. In some such instances, the conformationallyrigid sequence may sterically hinder the circular polyribonucleotidefrom binding a protein or a nucleic acid. In some embodiments, thesequence of the circular polyribonucleotide is purposefully engineeredto include such secondary structures to avoid or reduce protein ornucleic acid binding.

There are 16 possible base-pairings, however of these, six (AU, GU, GC,UA, UG, CG) may form actual base-pairs. The rest are called mismatchesand occur at very low frequencies in helices. In some embodiments, thestructure of the circular polyribonucleotide cannot easily be disruptedwithout impact on its function and lethal consequences, which provide aselection to maintain the secondary structure. In some embodiments, theprimary structure of the stems (i.e., their nucleotide sequence) canstill vary, while still maintaining helical regions. The nature of thebases is secondary to the higher structure, and substitutions arepossible as long as they preserve the secondary structure. In someembodiments, the circular polyribonucleotide has a quasi-helicalstructure. In some embodiments, the circular polyribonucleotide has atleast one segment with a quasi-helical structure. In some embodiments, asegment having a quasi-helical structure has at least 3, 4, 5, 6, 7, 8,9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26,27, 28, 29, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100,or more nucleotides. In some embodiments, the circularpolyribonucleotide has one or more segments (e.g., 2, 3, 4, 5, 6, ormore) having a quasi-helical structure. In some embodiments, thesegments are separated by 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15,16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45,50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, or more nucleotides. Insome embodiments, the circular polyribonucleotide includes at least oneof a U-rich or A-rich sequence or a combination thereof. In someembodiments, the U-rich and/or A-rich sequences are arranged in a mannerthat would produce a triple quasi-helix structure. In some embodiments,the circular polyribonucleotide has a double quasi-helical structure. Insome embodiments, the circular polyribonucleotide has one or moresegments (e.g., 2, 3, 4, 5, 6, or more) having a double quasi-helicalstructure. In some embodiments, the circular polyribonucleotide includesat least one of a C-rich and/or G-rich sequence. In some embodiments,the C-rich and/or G-rich sequences are arranged in a manner that wouldproduce triple quasi-helix structure. In some embodiments, the circularpolyribonucleotide has an intramolecular triple quasi-helix structurethat aids in stabilization.

In some embodiments, the circular polyribonucleotide has twoquasi-helical structure (e.g., separated by a phosphodiester linkage),such that their terminal base pairs stack, and the quasi-helicalstructures become colinear, resulting in a “coaxially stacked”substructure.

In some embodiments, the circular polyribonucleotide comprises atertiary structure with one or more motifs, e.g., a pseudoknot, ag-quadruplex, a helix, and coaxial stacking.

In some embodiments, the circular polyribonucleotide has at least onebinding site, e.g., at least one protein binding site, at least onemiRNA binding site, at least one lncRNA binding site, at least one tRNAbinding site, at least one rRNA binding site, at least one snRNA bindingsite, at least one siRNA binding site, at least one piRNA binding site,at least one snoRNA binding site, at least one snRNA binding site, atleast one exRNA binding site, at least one scaRNA binding site, at leastone Y RNA binding site, at least one hnRNA binding site, and/or at leastone tRNA motif.

Delivery

The circular polyribonucleotide described herein may also be included inpharmaceutical compositions with a delivery carrier.

Pharmaceutical compositions described herein may be formulates forexample including a carrier, such as a pharmaceutical carrier and/or apolymeric carrier, e.g., a liposome, and delivered by known methods to asubject in need thereof (e.g., a human or non-human agricultural ordomestic animal, e.g., cattle, dog, cat, horse, poultry). Such methodsinclude, but not limited to, transfection (e.g., lipid-mediated,cationic polymers, calcium phosphate, dendrimers); electroporation orother methods of membrane disruption (e.g., nucleofection), viraldelivery (e.g., lentivirus, retrovirus, adenovirus, AAV),microinjection, microprojectile bombardment (“gene gun”), fugene, directsonic loading, cell squeezing, optical transfection, protoplast fusion,impalefection, magnetofection, exosome-mediated transfer, lipidnanoparticle-mediated transfer, and any combination thereof. Methods ofdelivery are also described, e.g., in Gori et al., Delivery andSpecificity of CRISPR/Cas9 Genome Editing Technologies for Human GeneTherapy. Human Gene Therapy. July 2015, 26(7): 443-451.doi:10.1089/hum.2015.074; and Zuris et al. Cationic lipid-mediateddelivery of proteins enables efficient protein-based genome editing invitro and in vivo. Nat Biotechnol. 2014 Oct. 30; 33(1):73-80.

The invention is further directed to a host or host cell comprising thecircular polyribonucleotide described herein. In some embodiments, thehost or host cell is a plant, insect, bacteria, fungus, vertebrate,mammal (e.g., human), or other organism or cell.

In some embodiments, the circular polyribonucleotide is non-immunogenicin the host. In some embodiments, the circular polyribonucleotide has adecreased or fails to produce a response by the host's immune system ascompared to the response triggered by a reference compound, e.g. alinear polynucleotide corresponding to the described circularpolyribonucleotide or a circular polyribonucleotide lacking anencryptogen. Some immune responses include, but are not limited to,humoral immune responses (e.g. production of antigen-specificantibodies) and cell-mediated immune responses (e.g. lymphocyteproliferation).

In some embodiments, a host or a host cell is contacted with (e.g.,delivered to or administered to) the circular polyribonucleotide. Insome embodiments, the host is a mammal, such as a human. The amount ofthe circular polyribonucleotide, expression product, or both in the hostcan be measured at any time after administration. In certainembodiments, a time course of host growth in a culture is determined. Ifthe growth is increased or reduced in the presence of the circularpolyribonucleotide, the circular polyribonucleotide or expressionproduct or both is identified as being effective in increasing orreducing the growth of the host.

Methods of Production

In some embodiments, the circular polyribonucleotide includes adeoxyribonucleic acid sequence that is non-naturally occurring and canbe produced using recombinant technology (methods described in detailbelow; e.g., derived in vitro using a DNA plasmid) or chemicalsynthesis.

It is within the scope of the invention that a DNA molecule used toproduce an RNA circle can comprise a DNA sequence of anaturally-occurring original nucleic acid sequence, a modified versionthereof, or a DNA sequence encoding a synthetic polypeptide not normallyfound in nature (e.g., chimeric molecules or fusion proteins). DNA andRNA molecules can be modified using a variety of techniques including,but not limited to, classic mutagenesis techniques and recombinanttechniques, such as site-directed mutagenesis, chemical treatment of anucleic acid molecule to induce mutations, restriction enzyme cleavageof a nucleic acid fragment, ligation of nucleic acid fragments,polymerase chain reaction (PCR) amplification and/or mutagenesis ofselected regions of a nucleic acid sequence, synthesis ofoligonucleotide mixtures and ligation of mixture groups to “build” amixture of nucleic acid molecules and combinations thereof.

The circular polyribonucleotide may be prepared according to anyavailable technique including, but not limited to chemical synthesis andenzymatic synthesis. In some embodiments, a linear primary construct orlinear mRNA may be cyclized, or concatemerized to create a circularpolyribonucleotide described herein. The mechanism of cyclization orconcatemerization may occur through methods such as, but not limited to,chemical, enzymatic, splint ligation), or ribozyme catalyzed methods.The newly formed 5′-/3′-linkage may be an intramolecular linkage or anintermolecular linkage.

Methods of making the circular polyribonucleotides described herein aredescribed in, for example, Khudyakov & Fields, Artificial DNA: Methodsand Applications, CRC Press (2002); in Zhao, Synthetic Biology: Toolsand Applications, (First Edition), Academic Press (2013); and Egli &Herdewijn, Chemistry and Biology of Artificial Nucleic Acids, (FirstEdition), Wiley-VCH (2012).

Various methods of synthesizing circular polyribonucleotides are alsodescribed in the art (see, e.g., U.S. Pat. Nos. 6,210,931, 5,773,244,5,766,903, 5,712,128, 5,426,180, US Publication No. US20100137407,International Publication No. WO1992001813 and International PublicationNo. WO2010084371; the contents of each of which are herein incorporatedby reference in their entireties).

In some embodiments, the circular polyribonucleotides may be cleaned upafter production to remove production impurities, e.g., free ribonucleicacids, linear or nicked RNA, DNA, proteins, etc. In some embodiments,the circular polyribonucleotides may be purified by any known methodcommonly used in the art. Examples of nonlimiting purification methodsinclude, column chromatography, gel excision, size exclusion, etc.

Pharmaceutical Compositions

The present invention includes compositions in combination with one ormore pharmaceutically acceptable excipients. Pharmaceutical compositionsmay optionally comprise one or more additional active substances, e.g.therapeutically and/or prophylactically active substances.Pharmaceutical compositions of the present invention may be sterileand/or pyrogen-free. General considerations in the formulation and/ormanufacture of pharmaceutical agents may be found, for example, inRemington: The Science and Practice of Pharmacy 21st ed., LippincottWilliams & Wilkins, 2005 (incorporated herein by reference).

Although the descriptions of pharmaceutical compositions provided hereinare principally directed to pharmaceutical compositions which aresuitable for administration to humans, it will be understood by theskilled artisan that such compositions are generally suitable foradministration to any other animal, e.g., to non-human animals, e.g.non-human mammals. Modification of pharmaceutical compositions suitablefor administration to humans in order to render the compositionssuitable for administration to various animals is well understood, andthe ordinarily skilled veterinary pharmacologist can design and/orperform such modification with merely ordinary, if any, experimentation.Subjects to which administration of the pharmaceutical compositions iscontemplated include, but are not limited to, humans and/or otherprimates; mammals, including commercially relevant mammals such ascattle, pigs, horses, sheep, cats, dogs, mice, and/or rats; and/orbirds, including commercially relevant birds such as poultry, chickens,ducks, geese, and/or turkeys.

Formulations of the pharmaceutical compositions described herein may beprepared by any method known or hereafter developed in the art ofpharmacology. In general, such preparatory methods include the step ofbringing the active ingredient into association with an excipient and/orone or more other accessory ingredients, and then, if necessary and/ordesirable, dividing, shaping and/or packaging the product.

Methods of Expression

The present invention includes a method for protein expression,comprising translating at least a region of the circularpolyribonucleotide provided herein.

In some embodiments, the methods for protein expression comprisestranslation of at least 10%, at least 20%, at least 30%, at least 40%,at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, orat least 95% of the total length of the circular polyribonucleotide intopolypeptides. In some embodiments, the methods for protein expressioncomprises translation of the circular polyribonucleotide intopolypeptides of at least 5 amino acids, at least 10 amino acids, atleast 15 amino acids, at least 20 amino acids, at least 50 amino acids,at least 100 amino acids, at least 150 amino acids, at least 200 aminoacids, at least 250 amino acids, at least 300 amino acids, at least 400amino acids, at least 500 amino acids, at least 600 amino acids, atleast 700 amino acids, at least 800 amino acids, at least 900 aminoacids, or at least 1000 amino acids. In some embodiments, the methodsfor protein expression comprises translation of the circularpolyribonucleotide into polypeptides of about 5 amino acids, about 10amino acids, about 15 amino acids, about 20 amino acids, about 50 aminoacids, about 100 amino acids, about 150 amino acids, about 200 aminoacids, about 250 amino acids, about 300 amino acids, about 400 aminoacids, about 500 amino acids, about 600 amino acids, about 700 aminoacids, about 800 amino acids, about 900 amino acids, or about 1000 aminoacids. In some embodiments, the methods comprise translation of thecircular polyribonucleotide into continuous polypeptides as providedherein, discrete polypeptides as provided herein, or both.

In some embodiments, the translation of the at least a region of thecircular polyribonucleotide takes place in vitro, such as rabbitreticulocyte lysate. In some embodiments, the translation of the atleast a region of the circular polyribonucleotide takes place in vivo,for instance, after transfection of a eukaryotic cell, or transformationof a prokaryotic cell such as a bacteria.

In some aspects, the present disclosure provides methods of in vivoexpression of one or more expression sequences in a subject, comprising:administering a circular polyribonucleotide to a cell of the subjectwherein the circular polyribonucleotide comprises the one or moreexpression sequences; and expressing the one or more expressionsequences from the circular polyribonucleotide in the cell. In someembodiments, the circular polyribonucleotide is configured such thatexpression of the one or more expression sequences in the cell at alater time point is equal to or higher than an earlier time point. Insome embodiments, the circular polyribonucleotide is configured suchthat expression of the one or more expression sequences in the cell overa time period of at least 7, 8, 9, 10, 12, 14, 16, 18, 20, 22, 23 ormore days does not decrease by greater than about 40%. In someembodiments, the circular polyribonucleotide is configured such thatexpression of the one or more expression sequences in the cell ismaintained at a level that does not vary by more than about 40% for atleast 7, 8, 9, 10, 12, 14, 16, 18, 20, 22, 23 or more days. In someembodiments, the administration of the circular polyribonucleotide isconducted using any delivery method described herein. In someembodiments, the circular polyribonucleotide is administered to thesubject via intravenous injection. In some embodiments, theadministration of the circular polyribonucleotide includes, but is notlimited to, prenatal administration, neonatal administration, postnataladministration, oral, by injection (e.g., intravenous, intraarterial,intraperotoneal, intradermal, subcutaneous and intramuscular), byophthalmic administration and by intranasal administration.

In some embodiments, the methods for protein expression comprisemodification, folding, or other post-translation modification of thetranslation product. In some embodiments, the methods for proteinexpression comprise post-translation modification in vivo, e.g., viacellular machinery.

All references and publications cited herein are hereby incorporated byreference.

The above described embodiments can be combined to achieve theafore-mentioned functional characteristics. This is also illustrated bythe below examples which set forth exemplary combinations and functionalcharacteristics achieved. Table 1 provides an exemplary overview whichshows how different elements described above can be combined and thefunctional characteristics observed.

TABLE 1 Exemplary Elements in EXAMPLES Elements (e.g. start codon,stagger element, encryptogen, IRES etc.) Quasi-double strand ReplicationExpression Stagger Regulatory secondary element sequence element elementEncryptogen structure Exemplary function Ribosomal pausing; ModulatingTranscription Coding for rolling circle Expression immune Effect ofCircular start product translation modifier response PolyribonucleotideExample 3 x x x Greater translation efficiency than a linear counterpartExample 4 x x x Stochiometric translation efficiency of mutlipletranslation products Example 5 x x Less Example 9 immunogenicity Example44 than counterpart Example 47 lacking an encryptogen Example 13 xExample 14 x x x Increased half- life over a linear counterpart Example15 x x x Persistence during cell division Example 18 x x Increased half-Example 29 life over a linear counterpart Example 30 x x x Increasedhalf- life over a linear counterpart Example 38 x x x Greater Example 39translation efficiency than a linear counterpart Example 10 x Example 12Example 40 Example 41 Example 48 x Persistence during cell divisionExample 49 x x Greater translation efficiency than a linear counterpartExample 6 x x Example 52 Example 53 x x Less immunogenicity thancounterpart lacking an encryptogen Example 54 x x Greater Example 55translation efficiency than a linear counterpart; Increased half- lifeover a linear counterpart; Less immunogenicity than counterpart lackingan encryptogen

EXAMPLES

The following examples are provided to further illustrate someembodiments of the present invention, but are not intended to limit thescope of the invention; it will be understood by their exemplary naturethat other procedures, methodologies, or techniques known to thoseskilled in the art may alternatively be used.

Example 1: In Vitro Circular RNA Production

This example demonstrates in vitro production of a circular RNA.

A circular RNA is designed with a start-codon (SEQ ID NO:1), ORF(s) (SEQID NO:2), stagger element(s) (SEQ ID NOS 3, 132, and 133),encryptogen(s) (SEQ ID NOS 4 and 134), and an IRES (SEQ ID NO:5), shownin FIG. 2. Circularization enables rolling circle translation, multipleopen reading frames (ORFs) with alternating stagger elements fordiscrete ORF expression and controlled protein stoichiometry,encryptogen(s) to attenuate or mitigate RNA immunogenicity, and anoptional IRES that targets RNA for ribosomal entry without poly-Asequence.

In this Example, the circular RNA is generated as follows. Unmodifiedlinear RNA is synthesized by in vitro transcription using T7 RNApolymerase from a DNA segment having 5′- and 3′-ZKSCAN1 introns and anORF encoding GFP linked to 2A sequences. Transcribed RNA is purifiedwith an RNA purification system (QIAGEN), treated with alkalinephosphatase (ThermoFisher Scientific, EF0652) following themanufacturer's instructions, and purified again with the RNApurification system.

Splint ligation circular RNA is generated by treatment of thetranscribed linear RNA and a DNA splint using T4 DNA ligase (New EnglandBio, Inc., M0202M), and the circular RNA is isolated followingenrichment with RNase R treatment. RNA quality is assessed by agarosegel or through automated electrophoresis (Agilent).

Example 2: In Vivo Circular RNA Production, Cell Culture

This example demonstrates in vivo production of a circular RNA.

GFP (SEQ ID NO: 2) is cloned into an expression vector, e.g. pcDNA3.1(+)(Addgene) (SEQ ID NO: 6). This vector is mutagenized to induce circularRNA production in cells (SEQ ID NO: 6 and described by Kramer et al2015), shown in FIG. 3.

HeLa cells are grown at 37° C. and 5% CO₂ in Dulbecco's modified Eagle'smedium (DMEM) with high glucose (Life Technologies), supplemented withpenicillin—streptomycin and 10% fetal bovine serum. One microgram of theabove described expression plasmid is transfected using lipidtransfection reagent (Life Technologies), and total RNA from thetransfected cells is isolated using a phenol-based RNA isolation reagent(Life Technologies) as per the manufacturer's instructions between 1hour and 20 days after transfection.

To measure GFP circular RNA and mRNA levels, qPCR reverse transcriptionusing random hexamers is performed. In short, for RT-qPCR Hela cells'total RNA and RNase R-digested RNA from the same source are used astemplates for the RT-PCR. To prepare the cDNAs of GFP mRNAs and circularGFP RNAs, the reverse transcription reactions are performed with areverse transcriptase (Super-Script II: RNase H; Invitrogen) and randomhexamers in accordance with the manufacturer's instruction. Theamplified PCR products are analyzed using a 6% PAGE and visualized byethidium bromide staining. To estimate the enrichment factor, the PCRproducts are quantified by densitometry (ImageQuant; Molecular Dynamics)and the concentrations of total RNA samples are measured by UVabsorbance.

An additional RNA measurement is performed with northern blot analysis.Briefly, whole cell extract was obtained using a phenol based reagent(TRIzol) or nuclear and cytoplasmic protein extracts are obtained byfractionation of the cells with a commercial kit (CelLytic NuCLEARExtraction Kit, Sigma). To inhibit RNA polymerase II transcription,cells are treated with flavopiridol (1 mM final concentration; Sigma)for 0-6 h at 37° C. For RNase R treatments, 10 mg of total RNA istreated with 20 U of RNase R (Epicentre) for 1 h at 37° C.

Northern blots using oligonucleotide probes are performed as follows.Oligonucleotide probes, PCR primers are designed using standard primerdesigning tools. T7 promoter sequence is added to the reverse primer toobtain an antisense probe in in vitro transcription reaction. In vitrotranscription is performed using T7 RNA polymerase with a DIG-RNAlabeling mix according to manufacturer's instruction. DNA templates areremoved by DNAs I digestion and RNA probes purified by phenol chloroformextraction and subsequent precipitation. Probes are used at 50 ng/ml.Total RNA (2 μg-10 μg) is denatured using Glyoxal load dye (Ambion) andresolved on 1.2% agarose gel in MOPS buffer. The gel is soaked in 1×TBEfor 20 min and transferred to a Hybond-N+ membrane (GE Healthcare) for 1h (15 V) using a semi-dry blotting system (Bio-Rad). Membranes are driedand UV-crosslinked (at 265 nm) 1× at 120,000 μJ cm-2. Pre-hybridizationis done at 68° C. for 1 h and DIG-labelled in-vitro transcribed RNAprobes are hybridized overnight. The membranes are washed three times in2×SSC, 0.1% SDS at 68° C. for 30 min, followed by three 30 min washes in0.2×SSC, 0.1% SDS at 68° C. The immunodetection is performed withanti-DIG directly-conjugated with alkaline phosphatase antibodies.Immunoreactive bands are visualized using chemiluminescent alkalinephosphatase substrate (CDP star reagent) and an image detection andquantification system (LAS-4000 detection system).

Example 3: Preparation of Circular RNA and In Vitro Translation

This example demonstrates gene expression and detection of the geneproduct from a circular RNA.

In this Example, the circular RNA is designed with a start-codon (SEQ IDNO:1), a GFP ORF (SEQ ID NO:2), stagger element(s) (SEQ ID NOS 3, 132,and 133), human-derived encryptogen(s) (SEQ ID NOS 4 and 134), and withor without an IRES (SEQ ID NO:5), see FIG. 4. In this Example, thecircular RNA is generated either in vitro or in cells as described inExample 1 and 2.

The circular RNA is incubated for 5 h or overnight in rabbitreticulocyte lysate (Promega, Fitchburg, Wis., USA) at 30° C. The finalcomposition of the reaction mixture includes 70% rabbit reticulocytelysate, 10 μM methionine and leucine, 20 μM amino acids other thanmethionine and leucine, and 0.8 U/μL RNase inhibitor (Toyobo, Osaka,Japan). Aliquots are taken from the mixture and separated on 10-20%gradient polyacrylamide/sodium dodecyl sulfate (SDS) gels (Atto, Tokyo,Japan). The supernatant is removed and the pellet is dissolved in 2×SDSsample buffer (0.125 M Tris-HCl, pH 6.8, 4% SDS, 30% glycerol, 5%2-mercaptoethanol, 0.01% bromophenol blue) at 70° C. for 15 min. Thehemoglobin protein is removed during this process whereas proteins otherthan hemoglobin are concentrated.

After centrifugation at 1,400×g for 5 min, the supernatant is analyzedon 10-20% gradient polyacrylamide/SDS gels. A commercially availablestandard (BioRad) is used as the size marker. After beingelectrotransferred to a polyvinylidene fluoride (PVDF) membrane(Millipore) using a semi-dry method, the blot is visualized using achemiluminescent kit (Rockland).

It is expected that the GFP protein is visualized in cell lysates and isdetected in higher quantities in circular RNA than linear RNA, as aresult of rolling circle translation.

Example 4: Stoichiometric Protein Expression from Circular RNA

This example demonstrates the ability of circular RNA tostoichiometrically express of proteins.

In this Example, one circular RNA is designed to include encryptogens(SEQ ID NOS 4 and 134) and an ORF encoding GFP (SEQ ID NO: 2) and an ORFencoding RFP (SEQ ID NO:8) with stagger elements (SEQ ID NOS 3, 132, and133) flanking the GFP and RFP ORFs, see FIG. 5. Another circular RNA isdesigned similarly, however instead of flanking 2A sequences it willhave a Stop and Start codon in between the GFP and RFP ORFs. Thecircular RNAs are generated either in vitro or in cells as described inExample 1 and 2.

The circular RNAs are incubated for 5 h or overnight in rabbitreticulocyte lysate (Promega, Fitchburg, Wis., USA) at 30° C. The finalcomposition of the reaction mixture includes 70% rabbit reticulocytelysate, 10 μM methionine and leucine, 20 μM amino acids other thanmethionine and leucine, and 0.8 U/μL RNase inhibitor (Toyobo, Osaka,Japan). Aliquots are taken from the mixture and separated on 10-20%gradient polyacrylamide/sodium dodecyl sulfate (SDS) gels (Atto, Tokyo,Japan). The supernatant is removed and the pellet is dissolved in 2×SDSsample buffer (0.125 M Tris-HCl, pH 6.8, 4% SDS, 30% glycerol, 5%2-mercaptoethanol, 0.01% bromophenol blue) at 70° C. for 15 min. Thehemoglobin protein is removed during this process whereas proteins otherthan hemoglobin are concentrated.

After centrifugation at 1,400×g for 5 min, the supernatant is analyzedon 10-20% gradient polyacrylamide/SDS gels. A commercially availablestandard (BioRad) is used as the size marker. After beingelectrotransferred to a polyvinylidene fluoride (PVDF) membrane(Millipore) using a semi-dry method, the blot is visualized using achemiluminescent kit (Rockland).

It is expected that circular RNA with GFP and RFP ORFs not separated bya Stop and start codon will have equal amounts of either protein, whilecells treated with the circular RNA including the start and stop codonin between the ORFs will have different amounts of either protein.

Example 5: Non-Immunogenicity in Cell Culture

This example demonstrates in vivo assessment of immunogenicity of thecircular RNA after cell infection.

In this Example, circular RNAs designed to include an encryptogen e.g. aZKSCAN1 intron and a GFP ORF. In addition, control circular RNA isdesigned to include a GFP ORF with and without introns, see FIG. 6. Thecircular RNA is generated either in vitro or in cells as described inExample 1 and 2. HeLa cells are transfected with 500 ng of circularRNAs.

Transfection of the circular RNA include the following conditions: (1)naked circular RNA in cell culture media (Lingor et al 2004); (2)electroporation (Muller et al 2015); (3) cationic lipids (SNALP,Vaxfectin) (Chesnoy and Huang, 2000); (3) cationic polymers (PEI,polybrene, DEAE-dextran) (Turbofect); (4) virus-like particles (L1 fromHPV, VP1 from polyomavirus) (Tonges et al 2006); (5) exosomes (Exo-Fectfrom SBI); (6) nanostructured calcium phosphate (nanoCaP)(Olton et al2006); (6) peptide transduction domains (TAT, polyR,SP, pVEC, SynB1,etc) (Zhang et al 2009); (7) vesicles (VSV-G, TAMEL) (Liu et al 2017);(8) cell squeezing; (SQZ Biotechnologies) (9) nanoparticles (Neuhaus etal 2016); and/or (10) magnetofection (Mair et al 2009). Transfectionmethods are performed in cell culture media (DMEM 10% FBS) and cells aresubsequently cultured for 24-48 hrs.

After 2-48 hrs post-transfection, media is removed and relativeexpression of the indicated RNA and transfected RNA is measured byqRT-PCR.

For qRT-PCR analysis, total RNA is isolated from cells using a phenolbased RNA isolation solution (TRIzol) and an RNA isolation kit (QIAGEN)following the manufacturer's instructions. qRT-PCR analysis is performedin triplicate using a PCR master mix (Brilliant II SYBR Green qRT-PCRMaster Mix) and a PCR cycler (LightCycler 480). mRNA levels forwell-known innate immunity regulators such as RIG-I, MDAS, OAS, OASL,and PKR are quantified and normalized to actin, GAPDH, or HPRT values.Relative expression of indicated RNA genes for circular RNA transfectionare normalized by level of transfected RNA and compared to theexpression level of cells with circular RNA transfection that does notcontain encryptogen(s).

In addition to qRT-PCR analysis, western blot analysis andimmuno-histochemistry are used, as described above in Example 4, toassess GFP expression efficiency.

It is expected that GFP positive cells containing encryptogen(s) willshow an attenuated immunogenicity response.

In addition, (1) primary murine dendritic cells; (2) Human embryonickidney 293 cells stabile expressing TLR-7, 8 or 9 (InvivoGen); (3)monocyte derived dendritic cells (AllCells) or (4) Raw 264.7 cells aretransfected with a DNA plasmid including ZKSCAN1 or td introns thatproduce a circular RNA encoding GFP as described above. After 6-48 hrspost-transfection, cell culture supernatant is collected and cytokineexpression is measured using ELISA. When cell culture supernatant iscollected, cells are collected for Northern blot, gene expression arrayand FACS analysis.

For ELISA, ELISA kits for interferon-β (IFN-β), chemokine (C-C motif)ligand 5 (CCL5), IL-12 (BD Biosciences), IFN-α, TNF-α and IL-8(Biosource International) are used. ELISAs are performed according tothe manufacturers' recommendations. Expression of indicated cytokinesfor circular RNA transfected cells are compared to the level of controlRNA transfected cells. It is expected that cells transfected withcircular RNA with an encryptogen will have reduced cytokine expressioncompared to control transfected cells.

For Northern blot analysis. Samples are processed and analyzed aspreviously described. Probes are derived from plasmids and are specificfor the coding regions of human IFN-alpha 13, IFN-beta (OpenBiosystems), TNF-alpha, or GAPDH (ATCC). It is expected that cellstransfected with circular RNA with an encryptogen will have reducedcytokine expression compared to control transfected cells.

For the gene expression array, RNA is isolated using a phenol basedsolution (TRIzol) and/or an RNA isolation kit (RNeasy Qiagen). RNA isamplified and analyzed (e.g. Illumina Human HT12v4 chip in an IlluminaBeadStation 500GX). Levels in mock control treated cells are used as thebaseline for the calculation of fold increase. It is expected that cellstransfected with circular RNA with an encryptogen will have reducedcytokine expression compared to control transfected cells.

For FACS analysis, cells are stained with a directly conjugatedantibodies against CD83 (Research Diagnostics Inc), HLA-DR, CD80 or CD86and analyzed on a flow cytometer. It is expected that cells transfectedwith circular RNA with an encryptogen will show reduced expression ofthese markers compared to control transfected cells.

Example 6: Riboswitches for Selective Expression

This example demonstrates the ability to control protein expression fromcircular RNA in vivo.

For this Example, circular RNAs designed to include encryptogen(s) (SEQID NOS 4 and 134), a synthetic riboswitch (SEQ ID NO: 9) regulating theexpression of the ORF encoding GFP (SEQ ID NO:2) with stagger elements(2A sequences) (SEQ ID NOS 3, 132, and 133) flanking the GFP ORF, seeFIG. 7. The circular RNA is generated either in vitro or in cells asdescribed in Example 1 and 2.

Theophylline induces activation of the riboswitch, resulting in anoff-switch of gene expression (as described by Auslander et al 2010). Itis expected that the riboswitch controls GFP expression from thecircular RNA. In the presence of theophylline, no GFP expression isexpected to be observed.

HeLa cells are transfected with 500 ng of the described circular RNAencoding GFP under the control of the theophylline dependent syntheticriboswitch (SEQ ID NO:9) to assess selective expression. Transfectionmethods are described in Example 5.

After 24 hr of culture at 37° C. and 5% CO2, cells are treated with andwithout theophylline with concentrations ranging from 1 nM-3 mM. After24 hrs of continuous culture, cells are fixed in 4% paraformaldehyde for15 minutes at room temperature, blocked and permeabilized for 45 minuteswith 10% FBS in PBS with 0.2% detergent. Samples are then incubated withprimary antibodies against GFP (Invitrogen) and secondary antibodiesconjugated with Alexa 488 and DAPI (Invitrogen) in PBS with 10% FBS and0.1% detergent for 2 hrs at room temperature or overnight at 4° C. Cellsare then washed with PBS and subsequently analyzed using a fluorescentmicroscope for GFP expression.

Example 7: In Vivo Expression

This example demonstrates the ability to express protein from a circularRNA in vivo.

For this Example, circular RNAs designed to include includingencryptogen(s) (SEQ ID NOS 4 and 134) and an ORF encoding GFP (SEQ IDNO:2) or RFP (SEQ ID NO:8) or Luciferase (SEQ ID NO:10) with staggerelements (SEQ ID NOS 3, 132, and 133) flanking the GFP, RFP orLuciferase ORF, see FIG. 8. The circular RNA is generated either invitro or in cells as described in Example 1 and 2.

Male BALB/c mice 6-8 weeks old receive 300 mg/kg (6 mg) circular RNA (50uL vol) with GFP, RFP, or luciferase ORFs, as described herein, orlinear RNA as a control, via intradermal (ID), intramuscular (IM), oral(PO), intraperitoneal (IP), or intravenous (IV) administration. Animalsreceive a single dose or three injections (day 1, day 3, day 5).

Blood, heart, lung, spleen, kidney, liver, and skin injection sites arecollected from non-dosed control mice and at 2, 4, 8, 24, 48, 72, 96120, 168, and 264 hr post-dosing (n=4 mice/time point). Blood samplesare collected from jugular venipuncture at study termination.

Circular RNA quantification for both serum and tissues is performedusing quantification of branched DNA (bDNA) (Panomics/Affymetrix). Astandard curve on each plate of known amounts of RNA (added to untreatedtissue samples) is used to quantitate the RNA in treated tissues. Thecalculated amount in picograms (pg) is normalized to the amount ofweighed tissue in the lysate applied to the plate. Protein expression(RFP or GFP) is evaluated by FACS or western blot in each tissue asdescribed in a previous Example.

A separate group of mice dosed with luciferase circular RNA are injectedwith 3 mg luciferin at 6, 24, 48, 72, and 96 hr post-dosing and theanimals are imaged on an in vivo imaging system (IVIS Spectrum,PerkinElmer). At 6 hr post-dosing, three animals are sacrificed anddissected, and the muscle, skin, draining lymph nodes, liver, and spleenare imaged ex vivo.

It is expected that mice express GFP, RFP, or luciferase in treatedtissues.

Example 8: In Vivo Biodistribution

This example demonstrates the ability to control and measurebiodistribution of circular RNA in vivo.

In this Example, mice are treated with the circular RNA encodingluciferase as described in Example 9. In short, circular RNAs designedto include including encryptogen(s) (SEQ ID NOS 4 and 134) and an ORFencoding Luciferase (SEQ ID NO:10) with stagger elements (SEQ ID NO 3,132, and 133) flanking the Luciferase ORF, see FIG. 9. The circular RNAis generated either in vitro or in cells as described in Example 1 and2.

Mice are dosed with luciferase circular RNA by injected with 3 mgluciferin, at 6, 24, 48, 72, and 96 hr post-dosing and the animals areimaged on an in vivo imaging system (IVIS Spectrum, PerkinElmer). At 6hr post-dosing, three animals are sacrificed and dissected, and themuscle, skin, draining lymph nodes, liver, and spleen are imaged ex vivo

Circular RNA quantification for both serum and tissues is performed byusing quantification of branched DNA (bDNA) (Panomics/Affymetrix). Astandard curve on each plate of known amounts of RNA (added to untreatedtissue samples) is used to quantitate the RNA in treated tissues. Thecalculated amount in picograms (pg) is normalized to the amount ofweighed tissue in the lysate applied to the plate.

A separate group of male BALB/c mice 6-8 weeks old are dosed withluciferase circular RNA via IM or ID administration at four dose levels:10, 2, 0.4, and 0.08 mg (n=6 per group). At 6, 24, 48, 72, and 96 hrpost-dosing, animals are injected with 3 mg luciferin and imaged on anin vivo imaging system (IVIS Spectrum, PerkinElmer). At 6 hrpost-dosing, three animals are sacrificed and dissected, and the muscle,skin, draining lymph nodes, liver, and spleen are imaged ex vivo.Tissues from the mice are also assessed for luciferase expression asdescribed in Example 9 and tissue distribution of this expression isanalyzed.

It is expected that mice show expression of luciferase in the treatedtissues.

Example 9: Non-Immunogenicity In Vivo

This example demonstrates in vivo assessment of immunogenicity of thecircular RNA after cell infection.

This Example describes quantification and comparison of the immuneresponse after administrations of circular RNA harboring an encryptogen,see FIG. 10. In an embodiment, any of the circular RNA with anencryptogen, will have a reduced (e.g., reduced compared toadministration of control RNA) immunogenic response following one ormore administrations of the circular RNA compared to control.

A measure of immunogenicity for circular RNA are the cytokine levels inserum.

In this Example, cytokine serum levels are examined after one or moreadministrations of circular RNA. Circular RNA from any one of theprevious Examples is administered via intradermal (ID), intramuscular(IM), oral (PO), intraperitoneal (IP), or intravenous (IV) into BALB/cmice 6-8 weeks old. Serum is drawn from the different cohorts: miceinjected systemically and/or locally with injection(s) of circular RNAharboring an encryptogen and circular RNA without an encryptogen.

Collected serum samples are diluted 1-10× in PBS and analyzed for mouseIFN-α by enzyme-linked immunosorbent assay (PBL Biomedical Labs,Piscataway, N.J.) and TNF-α (R&D, Minneapolis, Minn.).

In addition to cytokine levels in serum, expression of inflammatorymarkers is another measure of immunogenicity. In this Example, spleentissue from mice treated with vehicle (no circular RNA), linear RNA, orcircular RNA will be harvested 1, 4, and 24 hours post administration.Samples will be analyzed using the following techniques qRT-PCRanalysis, Northern blot or FACS analysis.

For qRT-PCR analysis mRNA levels for RIG-I, MDA5, OAS, OASL, TNF-alphaand PKR are quantified as described previously.

For Northern blot analysis. Samples are processed and analyzed forIFN-alpha 13, IFN-beta (Open Biosystems), TNF-alpha, or GAPDH (ATCC) asdescribed above.

For FACS analysis, cells are stained with a directly conjugatedantibodies against CD83 (Research Diagnostics Inc), HLA-DR, CD80 or CD86and analyzed on a flow cytometer.

In an embodiment, circular RNA with an encryptogen will have decreasedcytokine levels (as measured by ELISA, Northern blot, FACS and/orqRT-PCR) after one or multiple administrations, as compared control RNA.

Example 10: Circular RNA Includes at Least One Double-Stranded RNASegment

This example demonstrates that circular RNA includes at least onedouble-stranded RNA segment.

In this Example, circular RNA is synthesized through one of the methodsdescribed previously, to include a GFP ORF and an IRES, see FIG. 11. Dotblot assays with J2 and K1 monoclonal antibodies will be utilized tomeasure double stranded RNA structures of at least 40 bp in length.Circular RNA (200 ng) is blotted onto a nylon membrane (super chargedNytran), dried, blocked with 5% non-fat dried milk in TBS-T buffer (50mM Tris-HCl, 150 mM NaCl, 0.05% Tween-20, pH 7.4), and incubated withdsRNA-specific mAb J2 or K1 (English & Scientific Consulting) for 60min. Membranes are washed six times with TBS-T then treated withHRP-conjugated donkey anti-mouse Ig (Jackson Immunology), then washedsix times and dots are visualized with an enhanced chemiluminescencewestern blot detection reagent (Amersham).

It is expected that a circular RNA creates an internal quasi-doublestranded RNA segment.

Example 11: Circular RNA Includes a Quasi-Double-Stranded Structure

This example demonstrates that circular RNA includes aquasi-double-stranded structure.

In this Example, circular RNA is synthesized through one of the methodsdescribed previously, with and without addition of the expression ofHDVmin (Griffin et al 2014). This RNA sequence forms a quasi-helicalstructure, see FIG. 12, and is used as a positive control (as shown byGriffin et al 2014).

To test if circular RNA structure includes a functionalquasi-double-stranded structure we will determine the secondarystructure using selective 2′OH acylation analyzed by primer extension(SHAPE). SHAPE assesses local backbone flexibility in RNA atsingle-nucleotide resolution. The reactivity of base positions to theSHAPE electrophile is related to secondary structure: base-pairedpositions are weakly reactive, while unpaired positions are more highlyreactive.

SHAPE is performed on circular RNA, HDVmin, and linear RNA containing.SHAPE is performed with N-methylisatoic anhydride (NMIA) or benzoylcyanide (BzCN) essentially as reported by Wilkinson et al 2006 andGriffin 2014 et al respectively. In brief for SHAPE with BzCN, 1 ul of800 mM BzCN in dimethyl sulfoxide (DMSO) is added to a 20 ul reactionmixture containing 3 to 6 pmol of RNA in 160 mM Tris, pH 8.0, 1 U/lRNAse inhibitor (e.g. SuperaseIn RNase inhibitor) and incubated for 1min at 37° C. Control reaction mixtures include 1 ul DMSO without BzCN.After incubation with BzCN, RNAs is extracted with phenol chloroform,and purified (e.g. using a RNA Clean & Concentrator-5 kit) as directedby the manufacturer, and resuspended in 6 ul 10 mM Tris, pH 8.0. Aone-dye system is used to detect BzCN adducts. RNAs are annealed with aprimer labeled with 6-carboxyfluorescein (6-FAM). Primer extension isperformed using a reverse transcriptase (SuperScript III—Invitrogen)according to the manufacturer's recommendations with the followingmodifications to the incubation conditions: 5 min at 42° C., 30 min at55° C., 25 min at 65° C., and 15 min at 75° C. Two sequencing laddersare generated using either 0.5 mM ddATP or 0.5 mM ddCTP in the primerextension reaction. Primer extension products are precipitated withethanol, washed to remove excess salt, and resolved by capillaryelectrophoresis along with a commercial size standard (e.g. Liz sizestandard Genewiz Fragment Analysis Service).

Raw electropherograms are analyzed using a primary fragment analysistool (e.g. PeakScanner Applied Bio-systems). The peaks at each positionin the electropherogram are then integrated. For each RNA analyzed, yaxis scaling to correct for loading error is performed so that thebackground for each primer extension reaction is normalized to that of anegative-control reaction performed on RNA that is not treated withBzCN. A signal decay correction is applied to the data for eachreaction. The peaks are aligned to a ladder created from two sequencingreactions. At each position, the peak area of the negative control issubtracted from the peak area in BzCN-treated samples; these values arethen converted to normalized SHAPE reactivities by dividing thesubtracted peak areas by the average of the highest 2% to 10% of thesubtracted peak areas.

In addition to SHAPE analysis we will perform NMR (Marchanka et al2015); Hydroxyl radical probing (Ding et al 2012); or a combination ofDMS and CMTC and Kethoxal (Tijerina et al 2007 and Ziehler et al 2001).

It is expected that a circular RNA will have a quasi-double-strandedstructure.

Example 12: Circular RNA Includes a Functional Quasi-Helical Structure

This example demonstrates that circular RNA includes a functionalquasi-helical structure.

In this Example, circular RNA is synthesized through one of the methodsdescribed previously, with the addition of the expression of 395L(Defenbaugh et al 2009). This RNA sequence forms a quasi-helicalstructure (as shown above, by RNA secondary structure folding algorithmmfold and Defenbaugh et al 2009), FIG. 13. This structure is essentialfor complex formation with hepatitis D antigen (HDAg).

Therefore, to test if circular RNA structure includes a functionalquasi-structure we will incubate circular RNA and linear RNA withHDAg-160 or HDAg-195 and analyze binding using EMSA assays. Bindingreactions are done in 25 ul including 10 mM Tris-HCl (pH 7.0), 25 mMKCl, 10 mM NaCl, 0.1 g/l bovine serum albumin (New England Biolabs), 5%glycerol, 0.5 mM DTT, 0.2 U/l RNase inhibitor (Applied Biosystems), and1 mM phenylmethylsulfonyl fluoride solution. circular RNA is incubatedwith HDAg protein (obtained as described by Defenbaugh et al 2009) atconcentrations ranging from 0-110 nM. Reaction mixtures are assembled onice, incubated at 37° C. for 1 h, and electrophoresed on 6% nativepolyacrylamide gels in 0.5 Tris-borate-EDTA at 240 V for 2.5 h. Levelsof free and bound RNA are determined using nucleic acid staining (e.g.gelred). Binding will be calculated as the intensity of unbound RNArelative to the intensity of the entire lane minus the background.

It is expected that a circular RNA will have a functional quasi-helicalstructure.

Example 13: Self-Transcription/Replication

In this Example, circular RNA is synthesized through one of the methodsdescribed previously, with the addition of the expression of the HDVreplication domain(s) (as described by Beeharry et al 2014), theantigenomic replication competent ribozyme and a nuclear localizationsignal. These RNA sequences allow for circular RNA to be located in thenucleus where the host RNA polymerase will bind and transcribe the RNA.Then this RNA is self-cleaved using the ribozyme. RNA is then ligatedand self-replicated again, see FIG. 14.

Circular RNA (1-5 microgram) will be transfected into HeLa cells usingtechniques described above. HeLa cells are grown at 37° C. and 5% CO₂ inDulbecco's modified Eagle's medium (DMEM) with high glucose (LifeTechnologies), supplemented with penicillin—streptomycin and 10% fetalbovine serum. After transfection HeLa cells are cultured for anadditional 4-72 hr, then total RNA from the transfected cells isisolated using a phenol-based RNA isolation reagent (Life Technologies)as per the manufacturer's instructions between 1 hour and 20 days aftertransfection and total amount of circular RNA encoding the HDV domainswill be determined and compared to control circular RNA using qPCR asdescribed herein.

Example 14: Circular RNA Stability/Half-Life

In this Example, circular RNA is synthesized through one of the methodsdescribed previously. A circular RNA is designed to include encryptogens(SEQ ID NOS 4 and 134) and an ORF encoding GFP (SEQ ID NO: 2) withstagger elements (SEQ ID NOS 3, 132, and 133) flanking the GFP ORF, seeFIG. 15.

Human fibroblast (e.g. IMR-90) are grown to confluency in Dulbecco'smodified Eagle's medium (DMEM; Invitrogen) supplemented with 10% fetalbovine serum (FBS; Invitrogen) at 37° C. under 5% CO2 on tissue culturetreated plates. When fibroblast reach confluency, they stop dividing dueto contact inhibition (Leontieva et al 2014). Lipid transfection reagent(2 μL; Invitrogen) is added to a mixture of 1 μg circular RNA or linearRNA (described above) and 145 μL reduced serum medium (Opti-MEM Isolution) in one well of a 12-well tissue culture treated plate. Afterincubation at room temperature for 15 min, ˜1×10{circumflex over ( )}5cells suspended in DMEM with 10% FBS are added to the circular RNAsolution (described above).

Cells will be cultured and then collected at day 1, 2, 3, 4, 5, 10, 20and 30 after circular RNA transfection. Cells will be isolated forq-rt-PCR and another subset for FACS analysis. To measure GFP circularRNA and mRNA levels, qPCR reverse transcription using random hexamers isperformed, as described in Example 2. Cells will be analyzed with FACSusing GFP antibodies as described herein.

It is expected that circular RNA will persist in cells for at leastseveral days and that they retain functional expression of GFP protein.

Example 15: Circular RNA Preservation in Daughter Cells

In this Example, circular RNA is synthesized through one of the methodsdescribed previously. A circular RNA is designed to include encryptogens(SEQ ID NO 4 and 134) and an ORF encoding GFP (SEQ ID NO: 2) withstagger elements (SEQ ID NOS 3, 132, and 133) flanking the GFP ORF, seeFIG. 16.

Human Fibroblasts (e.g. IMR-90) are grown in Dulbecco's modified Eagle'smedium (DMEM; Invitrogen) supplemented with 10% fetal bovine serum (FBS;Invitrogen) at 37° C. under 5% CO2 on tissue culture treated plates.Cells are passaged regularly to maintain exponential growth. Lipidtransfection reagent (2 μL; Invitrogen) is added to a mixture of 1 μgcircular RNA or linear RNA (described above) and 145 μL reduced serummedium (Opti-MEM I solution) in one well of a 12-well tissue culturetreated plate. After incubation at room temperature for 15 min,1×10{circumflex over ( )}5 HeLa cells suspended in DMEM with 10% FBS areadded to the circular RNA solution (described above). After incubationfor 24 h at 37° C. and 5% CO2, the cells are pulsed with BrdU (e.g.Sigma-Aldrich). BrdU, labeling duration is optimized for each cell typeaccording to their specific population doubling time, e.g. IMR-90 humanfibroblasts have a doubling time of 27 hrs and are pulsed for 8-9 hrs asdescribed by Elabd et al 2013.

Cells will be collected at day 1, 2, 3, 4, 5 and 10 after BrdU pulse. Asubset of the cells will be isolated q-rt-PCR and another subset forFACS analysis. To measure GFP circular RNA and mRNA levels, qPCR reversetranscription using random hexamers is performed, as described inExample 2. Cells will be analyzed with FACS using BrdU and GFPantibodies as described herein.

It is expected that circular RNA will persist in daughter cells and thatdaughter cells will express GFP protein.

Example 16: Circular RNA Circularization

This Example demonstrates in vitro production of circular RNA usingsplint ligation.

A non-naturally occurring circular RNA can be engineered to include oneor more desirable properties and may be produced using recombinant DNAtechnology. As shown in the following Example, splint ligationcircularized linear RNA.

CircRNA1 was designed to encode triple FLAG tagged EGF without stopcodon (264nts). It has a Kozak sequence (SEQ ID NO: 11) at the startcodon for translation initiation. CirRNA2 has identical sequences withcircular RNA1 except it has a termination element (triple stop codons)(273nts, SEQ ID NO: 12). Circular RNA3 was designed to encode tripleFLAG tagged EGF flanked by a stagger element (2A sequence, SEQ ID NO:13), without a termination element (stop codon) (330 nts). CircRNA4 hasidentical sequences with circular RNA3 except it has a terminationelement (triple stop codon) (339nts).

In this example, the circular RNA was generated as follows. DNAtemplates for in vitro transcription were amplified from gBlocks genefragment with corresponding sequences (IDT) with T7 promoter-harboringforward primer and 2-O-methylated nucleotide with a reverse primer.Amplified DNA templates were gel-purified with a DNA gel purificationkit (Qiagen). 250-500 ng of purified DNA template was subjected to invitro transcription. Linear, 5′-mono phosphorylated in vitro transcriptswere generated using T7 RNA polymerase from each DNA template havingcorresponding sequences in the presence of 7.5 mM GMP, 1.5 mM GTP, 7.5mM UTP, 7.5 mM CTP and 7.5 mM ATP. Around 40 μg of linear RNA wasgenerated in each reaction. After incubation, each reaction was treatedwith DNase to remove the DNA template. The in vitro transcribed RNA wasprecipitated with ethanol in the presence of 2.5M ammonium acetate toremove unincorporated monomers.

Transcribed linear RNA was circularized using T4 RNA ligase 2 on a 20ntsplint DNA oligomer (SEQ ID NO: 14) as template. Splint DNA was designedto anneal 10 nt of each 5′ or 3′end of linear RNA. After annealing withthe splint DNA (3 μM), 1 μM linear RNA was incubated with 0.5 U/μl T4RNA ligase 2 at 37 C or 4 hr. Mixture without T4 RNA ligase 2 was usedas the negative control.

The circularization of linear RNA was monitored by separating RNA on 6%denaturing PAGE. Slower migrating RNA bands correspond with circular RNArather than linear RNA on denaturing polyacrylamide gels because oftheir circular structure. As seen in FIG. 17, the addition of ligase(+lanes) to the RNA mixtures generated new bands to appear above thelinear RNA bands that were present in the mixtures that lacked ligase(−lanes). Slower migrating bands appeared in all RNA mixtures indicatingsuccessful splint ligation (e.g., circularization) occurred withmultiple constructs as compared to negative control.

Example 17: RNA Circularization Efficiency

This Example demonstrates circularization efficiencies of RNA splintligation.

A non-naturally occurring circular RNA engineered to include one or moredesirable properties may be produced using splint mediatedcircularization. As shown in the following Example, splint ligationcircularized linear RNA with higher efficiency than controls.

CircRNA1, CircRNA2, CircRNA3, and CircRNA4 as described in Example 1were also used here. CircRNA5 was designed to encode FLAG tagged EGFflanked by a 2A sequence and followed by FLAG tagged nano luciferase(873nts, SEQ ID NO: 17). CircRNA6 has identical sequence with circularRNA5 except it included a a termination element (triple stop codon)between the EGF and nano luciferase genes, and a termination element(triple stop codon) at the end of the nano luciferase sequence (762nts,SEQ ID NO: 18).

In this Example, to measure circularization efficiency of RNA, 6different sizes of linear RNA (264nts, 273nts, 330 nts, 339nts, 873ntsand 762nts) were generated and circularized as described in Example 1.The circular RNAs were resolved by 6% denaturing PAGE and correspondingRNA bands on the gel for linear or circular RNA were excised forpurification. Excised RNA gel bands were crushed and RNA was eluted with800 μl of 300 mM NaCl overnight. Gel debris was removed by centrifugefilters and RNA was precipitated with ethanol in the presence of 0.3Msodium acetate.

Circularization efficiency was calculated as follows. The amount ofeluted circular RNA was divided by the total eluted RNA amount(circular+linear RNA) and the result was depicted as a graph in FIG. 18.

Ligation of linear RNAs using T4 RNAse ligase 2 produced circular RNA atefficiency rates higher than control. Trending data indicated largerconstructs circularized at higher rates.

Example 18: Circular RNA Lacking Degradation Susceptibility

This Example demonstrates circular RNA susceptibility to degradation byRNAse R compared to linear RNA.

Circular RNA is more resistant to exonuclease degradation than linearRNA due to the lack of 5′ and 3′ ends. As shown in the followingExample, circular RNA was less susceptible to degradation than itslinear RNA counterpart.

CircRNA5 was generated and circularized as described in Example 2 foruse in the assay described herein.

To test circularization of CircRNA5, 20 ng/μl of linear or CircRNA5 wasincubated with 2 U/μl of RNAse R, a 3′ to 5′ exoribonuclease thatdigests linear RNAs but does not digest lariat or circular RNAstructures, at 37° C. for 30 min. After incubation, the reaction mixturewas analyzed by 6% denaturing PAGE.

The linear RNA bands present in the lanes lacking exonuclease wereabsent in the CircRNA5 lane (see FIG. 19) indicating CircRNA5 showedhigher resistance to exonuclease treatment as compared to linear RNAcontrol.

Example 19: Isolation and Purification of Circular RNA

This Example demonstrates circular RNA purification.

In certain embodiments, circular RNAs, as described in the previousExamples, may be isolated and purified before expression of the encodedprotein products. This Example describes isolation using UREA gelseparation. As shown in the following Example, circular RNA was isolatedand purified.

CircRNA1, CircRNA2, CircRNA3, CircRNA4, CircRNA5, and CircRNA6, asdescribed in Example 2, were isolated as described herein.

In this Example, linear and circular RNA were generated as described. Topurify the circular RNAs, ligation mixtures were resolved on 6%denaturing PAGE and RNA bands corresponding to each of the circular RNAswere excised. Excised RNA gel fragments were crushed and RNA was elutedwith 800 μl of 300 mM NaCl overnight. Gel debris was removed bycentrifuge filters and RNA was precipitated with ethanol in the presenceof 0.3M sodium acetate. Eluted circular RNA was analyzed by 6%denaturing PAGE, see FIG. 20.

Single bands were visualized by PAGE for the circular RNAs havingvariable sizes.

Example 20: Detection of Protein Expression

This Example demonstrates in vitro protein expression from a circularRNA.

Protein expression is the process of generating a specific protein frommRNA. This process includes the transcription of DNA into messenger RNA(mRNA), followed by the translation of mRNA into polypeptide chains,which are ultimately folded into functional proteins and may be targetedto specific subcellular or extracellular locations.

As shown in the following Example, a protein was expressed in vitro froma circular RNA sequence.

Circular RNA was designed to encode triple FLAG tagged EGF flanked by a2A sequence without a termination element (stop codon) (330 nts, SEQ IDNO: 19).

Linear or circular RNA was incubated for 5 hr in rabbit reticulocytelysate at 30° C. in a volume of 25 μl. The final composition of thereaction mixture contained 70% rabbit reticulocyte lysate, 20 μM aminoacids, 0.8 U/μl RNase inhibitor and 1 μg of linear or circular RNA.After incubation, hemoglobin protein was removed by adding acetic acid(0.32 μl) and water (300 μl) to the reaction mixture (16 μl) andcentrifuging at 20,817×g for 10 min at 15° C. The supernatant wasremoved and the pellet was dissolved in 30° l of 2×SDS sample buffer andincubated at 70° C. for 15 min. After centrifugation at 1400×g for 5min, the supernatant was analyzed on a 10-20% gradientpolyacrylamide/SDS gel.

After being electrotransferred to a nitrocellulose membrane using drytransfer method, the blot was incubated with an anti-FLAG antibody andanti-mouse IgG peroxidase. The blot was visualized with an ECL kit (seeFIG. 21) and western blot band intensity was measured by ImageJ.

Fluorescence was detected indicated expression product was present.Thus, circular RNA was shown to drive expression of a protein.

Example 21: IRES-Independent Expression

This Example demonstrates circular RNA driving expression in the absenceof an IRES.

An IRES, or internal ribosome entry site, is an RNA element that allowstranslation initiation in a cap-independent manner. Circular RNA wasshown to be drive expression of Flag protein in the absence of an IRES.

Circular RNA was designed to encode triple FLAG tagged EGF flanked by a2A sequence without a termination element (stop codon) (330 nts, SEQ IDNO: 19).

Linear or circular RNA was incubated for 5 hr in rabbit reticulocytelysate at 30° C. in a volume of 250. The final composition of thereaction mixture included 70% rabbit reticulocyte lysate, 20 μM aminoacids, 0.8 U/μl RNase inhibitor and 1 μg of linear or circular RNA.After incubation, hemoglobin protein was removed by adding acetic acid(0.32 μl) and water (300 μl) to the reaction mixture (16 μl) andcentrifuging at 20,817×g for 10 min at 15° C. The supernatant wasremoved and the pellet was dissolved in 30°l of 2×SDS sample buffer andincubated at 70° C. for 15 min. After centrifugation at 1400×g for 5min, the supernatant was analyzed on a 10-20% gradientpolyacrylamide/SDS gel.

After being electrotransferred to a nitrocellulose membrane using drytransfer method, the blot was incubated with an anti-FLAG antibody andanti-mouse IgG peroxidase. The blot was visualized with an enhancedchemiluminescence (ECL) kit (see FIG. 21) and western blot bandintensity was measured by ImageJ.

Expression product was detected in the circular RNA reaction mixtureeven in the absence of an IRES.

Example 22: Cap-Independent Expression

This Example demonstrates circular RNA is able to drive expression inthe absence of a cap.

A cap is a specially altered nucleotide on the 5′ end of mRNA. The 5′cap is useful for stability, as well as the translation initiation, oflinear mRNA. Circular RNA drove expression of product in the absence ofa cap.

Circular RNA was designed to encode triple FLAG tagged EGF flanked by a2A sequence without a termination element (stop codon) (330 nts, SEQ IDNO: 19).

Linear or circular RNA was incubated for 5 hr in rabbit reticulocytelysate at 30° C. in a volume of 25 μl. The final composition of thereaction mixture included 70% rabbit reticulocyte lysate, 20 μM aminoacids, 0.8 U/μl RNase inhibitor and 1 μg of linear or circular RNA.After incubation, hemoglobin protein was removed by adding acetic acid(0.32 μl) and water (300 μl) to the reaction mixture (16 μl) andcentrifuging at 20,817×g for 10 min at 15° C. The supernatant wasremoved and the pellet was dissolved in 30 μl of 2×SDS sample buffer at70° C. for 15 min. After centrifugation at 1400×g for 5 min, thesupernatant was analyzed on 10-20% gradient polyacrylamide/SDS gel.

After being electrotransferred to a nitrocellulose membrane using drytransfer method, the blot was incubated with an anti-FLAG antibody andanti-mouse IgG peroxidase. The blot was visualized with an ECL kit (seeFIG. 21) and western blot band intensity was measured by ImageJ.

Expression product was detected in the circular RNA reaction mixtureeven in the absence of a cap.

Example 23: Expression Without a 5′-UTR

This Example demonstrates in vitro protein expression from a circularRNA lacking 5′ untranslated regions.

The 5′ untranslated region (5′ UTR) is the region directly upstream ofan initiation codon that aids in downstream protein translation of a RNAtranscript.

As shown in the following Example, a 5′-untranslated region in thecircular RNA sequence was not necessary for in vitro protein expression.

Circular RNA was designed to encode triple FLAG tagged EGF flanked by a2A sequence without a termination element (stop codon) (330 nts, SEQ IDNO: 19).

Linear or circular RNA was incubated for 5 hr in rabbit reticulocytelysate at 30° C. in a volume of 25 μl. The final composition of thereaction mixture included 70% rabbit reticulocyte lysate, 20 μM aminoacids, 0.8 U/μl RNase inhibitor and 1 μg of linear or circular RNA.After incubation, hemoglobin protein was removed by adding acetic acid(0.32 μl) and water (300 μl) to the reaction mixture (16 μl) andcentrifuging at 20,817×g for 10 min at 15° C. The supernatant wasremoved and the pellet was dissolved in 30°l of 2×SDS sample buffer andincubated at 70° C. for 15 min. After centrifugation at 1400×g for 5min, the supernatant was analyzed on a 10-20% gradientpolyacrylamide/SDS gel.

After being electrotransferred to a nitrocellulose membrane using drytransfer method, the blot was incubated with an anti-FLAG antibody andanti-mouse IgG peroxidase. The blot was visualized with an ECL kit (seeFIG. 21) and western blot band intensity was measured by ImageJ.

Expression product was detected in the circular RNA reaction mixtureeven in the absence of a 5′ UTR.

Example 24: Expression Without a 3′-UTR

This Example demonstrates in vitro protein expression from a circularRNA lacking a 3′-UTR.

The 3′ untranslated region (3′-UTR) is the region directly downstream ofa translation termination codon and includes regulatory regions that maypost-transcriptionally influence gene expression. The 3′-untranslatedregion may also play a role in gene expression by influencing thelocalization, stability, export, and translation efficiency of an mRNA.In addition, the structural characteristics of the 3′-UTR as well as itsuse of alternative polyadenylation may play a role in gene expression.

As shown in the following Example, a 3′-UTR in the circular RNA sequencewas not necessary for in vitro protein expression.

Circular RNA was designed to encode triple FLAG tagged EGF flanked by a2A sequence without a termination element (stop codon) (330 nts, SEQ IDNO: 19).

Linear or circular RNA was incubated for 5 hr in rabbit reticulocytelysate at 30° C. in a volume of 250. The final composition of thereaction mixture included 70% rabbit reticulocyte lysate, 20 μM aminoacids, 0.8 U/μl RNase inhibitor and 1 μg of linear or circular RNA.After incubation, hemoglobin protein was removed by adding acetic acid(0.32 μl) and water (300 μl) to the reaction mixture (16 μl) andcentrifuging at 20,817×g for 10 min at 15° C. The supernatant wasremoved and the pellet was dissolved in 30°l of 2×SDS sample buffer andincubated at 70° C. for 15 min. After centrifugation at 1400×g for 5min, the supernatant was analyzed on a 10-20% gradientpolyacrylamide/SDS gel.

After being electrotransferred to a nitrocellulose membrane using drytransfer method, the blot was incubated with an anti-FLAG antibody andanti-mouse IgG peroxidase. The blot was visualized with an ECL kit (seeFIG. 21) and western blot band intensity was measured by ImageJ.

Expression product was detected in the circular RNA reaction mixtureeven in the absence of a 3′UTR.

Example 25: Expression without a Termination Codon

This Example demonstrates generation of a polypeptide product followingrolling circle translation from a circular RNA lacking a terminationcodon.

Proteins are based on polypeptides, which are comprised of uniquesequences of amino acids. Each amino acid is encoded in mRNA bynucleotide triplets called codon. During protein translation, each codonin mRNA corresponds to the addition of an amino acid in a growingpolypeptide chain. Termination element or stop codons signal thetermination of this process by binding release factors, which cause theribosomal subunits to disassociate, releasing the amino acid chain.

As shown in the following Example, a circular RNA lacking a terminationcodon generated a large polypeptide product comprised of repeatedpolypeptide sequences via rolling circle translation.

Circular RNA was designed to encode triple FLAG tagged EGF without atermination element (stop codon) (264nts, SEQ ID NO: 20), and included aKozak sequence at the start codon to favor translation initiation.

Linear or circular RNA was incubated for 5 hr in rabbit reticulocytelysate at 30° C. in a volume of 25 μl. The final composition of thereaction mixture included 70% rabbit reticulocyte lysate, 20 μM aminoacids, 0.8 U/μl RNase inhibitor and 1 μg of linear or circular RNA.After incubation, hemoglobin protein was removed by adding acetic acid(0.32 μl) and water (300 μl) to the reaction mixture (16 μl) andcentrifuging at 20,817×g for 10 min at 15° C. The supernatant wasremoved and the pellet was dissolved in 30°l of 2×SDS sample buffer andincubated at 70° C. for 15 min. After centrifugation at 1400×g for 5min, the supernatant was analyzed on a 10-20% gradientpolyacrylamide/SDS gel.

After being electrotransferred to a nitrocellulose membrane using drytransfer method, the blot was incubated with an anti-FLAG antibody andanti-mouse IgG peroxidase. The blot was visualized with an ECL kit (seeFIG. 22) and western blot band intensity was measured by ImageJ.

Expression product was detected in the circular RNA reaction mixtureeven in the absence of a termination codon.

Example 26: Expression of Discrete Proteins without a TerminationElement (Stop Codon)

This Example demonstrates generation of a discrete protein productstranslated from a circular RNA lacking a termination element (stopcodons).

Stagger elements, such as 2A peptides, may include short amino acidsequences, ˜20 aa, that allow for the production of equimolar levels ofmultiple genes from a single mRNA. The stagger element may function bymaking the ribosome skip the synthesis of a peptide bond at theC-terminus of the 2A element, leading to separation between the end ofthe 2A sequence and the next peptide downstream. The separation occursbetween Glycine and Proline residues found on the C-terminus and theupstream cistron has a few additional residues added to the end, whilethe downstream cistron starts with a Proline.

As shown in the following Example, the circular RNA lacking atermination element (stop codon) generated a large polypeptide polymer(FIG. 23 left panel: no stagger—circular RNA lane) and inclusion of a 2Asequence at the 3′ end of the coding region resulted in production ofdiscrete protein at a size comparable to that generated by theequivalent linear RNA construct (FIG. 23 right panel: stagger—circularRNA lane).

Circular RNA was designed to encode triple FLAG tagged EGF without atermination element (stop codon) (264 nts, SEQ ID NO: 20) and without astagger element. A second circular RNA was designed to encode tripleFLAG tagged EGF flanked by a 2A sequence without a termination element(stop codon) (330 nts, SEQ ID NO: 19).

Linear or circular RNA was incubated for 5 hr in rabbit reticulocytelysate at 30° C. in a volume of 25 μl. The final composition of thereaction mixture included 70% rabbit reticulocyte lysate, 20 μM aminoacids, 0.8 U/μl RNase inhibitor and 1 μg of linear or circular RNA.After incubation, hemoglobin protein was removed by adding acetic acid(0.32 μl) and water (300 μl) to the reaction mixture (16 μl) andcentrifuging at 20,817×g for 10 min at 15° C. The supernatant wasremoved and the pellet was dissolved in 30°l of 2×SDS sample buffer andincubated at 70° C. for 15 min. After centrifugation at 1400×g for 5min, the supernatant was analyzed on a 10-20% gradientpolyacrylamide/SDS gel.

After being electrotransferred to a nitrocellulose membrane using drytransfer method, the blot was incubated with an anti-FLAG antibody andanti-mouse IgG peroxidase. The blot was visualized with an ECL kit (seeFIG. 23) and western blot band intensity was measured by ImageJ.

Discrete expression products were detected indicating circular RNAcomprising a stagger element drove expression of the individual proteinseven in the absence of a termination element (stop codons).

Example 27: Rolling Circle Translation

This Example demonstrates elevated in vitro biosynthesis of a proteinfrom circular RNA using a stagger element.

A non-naturally occurring circular RNA was engineered to include astagger element to compare protein expression with circular RNA lackinga stagger element. As shown in the following Example, a stagger elementoverexpressed protein as compared to an otherwise identical circular RNAlacking such a sequence.

Circular RNA was designed to encode triple FLAG tagged EGF with atermination element (e.g., three stop codons in tandem) (273nts, SEQ IDNO: 21). A second circular RNA was designed to encode triple FLAG taggedEGF flanked by a 2A sequence without a termination element (stop codon)(330 nts, SEQ ID NO: 19).

Linear or circular RNA was incubated for 5 hr in rabbit reticulocytelysate at 30° C. in a volume of 25 μl. The final composition of thereaction mixture contained 70% rabbit reticulocyte lysate, 20 μM aminoacids, 0.8 U/μl RNase inhibitor and 1 μg of linear or circular RNA.After incubation, hemoglobin protein was removed by adding acetic acid(0.32 μl) and water (300 μl) to the reaction mixture (16 μl) andcentrifuging at 20,817×g for 10 min at 15° C. The supernatant wasremoved and the pellet was dissolved in 30°l of 2×SDS sample buffer andincubated at 70° C. for 15 min. After centrifugation at 1400×g for 5min, the supernatant was analyzed on a 10-20% gradientpolyacrylamide/SDS gel.

After being electrotransferred to a nitrocellulose membrane using drytransfer method, the blot was incubated with an anti-FLAG antibody andanti-mouse IgG peroxidase. The blot was visualized with an ECL kit (seeFIG. 24) and western blot band intensity was measured by ImageJ.

Discrete expression products were detected indicating circular RNAcomprising a stagger element drove expression of the individual proteinseven in the absence of a termination element (stop codons).

Example 28: Expression of a Biologically Active Protein In Vitro

This Example demonstrates in vitro biosynthesis of a biologically activeprotein from circular RNA.

A non-naturally occurring circular RNA was engineered to express abiologically active therapeutic protein. As shown in the followingExample, a biologically active protein was expressed from the circularRNA in reticulocyte lysate.

Circular RNA was designed to encode FLAG tagged EGF flanked by a 2Asequence and followed by FLAG tagged nano-luciferase (873nts, SEQ IDNO:17).

Linear or circular RNA was incubated for 5 hr in rabbit reticulocytelysate at 30° C. in a volume of 250. The final composition of thereaction mixture contained 70% rabbit reticulocyte lysate, 20 μM aminoacids, 0.8 U/μl RNase inhibitor. Luciferase activity in the translationmixture was monitored using a luciferase assay system according tomanufacturer's protocol (Promega).

As shown in FIG. 25, much higher fluorescence was detected with bothcircular RNA and linear RNA than the control vehicle RNA, indicatingexpression product was present. Thus, circular RNA was shown to expressa biologically active protein.

Example 29: Circular RNA with a Longer Half-Life than Linear RNACounterpart

This Example demonstrates circular RNA engineered to have a prolongedhalf-life as compared to a linear RNA.

Circular RNA encoding a therapeutic protein provided recipient cellswith the ability to produce greater levels of the encoded proteinstemming from a prolonged biological half-life, e.g., as compared tolinear RNA. As shown in the following Example, a circular RNA had agreater half-life than its linear RNA counterpart in reticulocytelysate.

A circular RNA was designed to encode FLAG tagged EGF flanked by a 2Asequence and followed by FLAG tagged nano luciferase (873nts, SEQ ID NO:17).

In this Example, a time-course experiment was performed to monitor RNAstability. 100 ng of linear or circular RNA was incubated with rabbitreticulocyte lysate and samples were collected at 1 hr, 5 hr, 18 hr and30 hr. Total RNA was isolated from lysate using a phenol-based reagent(Invitrogen) and cDNA was generated by reverse transcription. qRT-PCRanalysis was performed using a dy-based quantitative PCR reaction mix(BioRad).

As shown in FIG. 26, greater concentrations of circular RNA weredetected in the later timepoints than linear RNA. Thus, the circular RNAwas more stable or had an increased half-life as compared to its linearcounterpart.

Example 30: Circular RNA Demonstrated a Longer Half-Life than Linear RNAin Cells

This Example demonstrates circular RNA delivered into cells and has anincreased half-life in cells compared with linear RNA.

A non-naturally occurring circular RNA was engineered to express abiologically active therapeutic protein. As shown in the followingExample, circular RNA was present at higher levels compared to itslinear RNA counterpart, demonstrating a longer half-life for circularRNA.

In this Example, circular RNA and linear RNA were designed to encode aKozak, EGF, flanked by a 2A, a stop or no stop sequence (SEQ ID NOs: 11,19, 20, 21). To monitor half-life of RNA in cells, 0.1×10⁶ cells wereplated onto each well of a 12 well plate. After 1 day, 1 μg of linear orcircular RNA was transfected into each well using a lipid-basedtransfection reagent (Invitrogen). Twenty-four hours after transfection,total RNA was isolated from cells using a phenol-based extractionreagent (Invitrogen). Total RNA (500 ng) was subjected to reversetranscription to generate cDNA. qRT-PCR analysis was performed using adye-based quantitative PCR mix (BioRad). Primer sequences were asfollow: Primers for linear or circular RNA, F: ACGACGGTGTGTGCATGTAT (SEQID NO: 106), R: TTCCCACCACTTCAGGTCTC (SEQ ID NO: 107); primers forcircular RNA, F: TACGCCTGCAACTGTGTTGT (SEQ ID NO: 108), R:TCGATGATCTTGTCGTCGTC (SEQ ID NO: 109).

Circular RNA was successfully transfected into 293T cells, as was itslinear counterpart. After 24 hours, the circular and linear RNA thatremained were measured using qPCR. As shown in FIGS. 27A and B, circularRNA was shown to have a longer half-life in cells compared to linearRNA.

Example 31: Synthetic Circular RNA were Translated in Cells, andSynthetic Circular RNA was Translated Via Rolling Circle Translation

This Example demonstrates translation of synthetic circular RNA incells.

As shown in the following Example, circular RNA and linear RNA weredesigned to encode a Kozak, 3×FLAG-EGF sequence with no terminationelement (SEQ ID NO: 11). Circular RNA was translated into polymeric EGF,while linear RNA was not, demonstrating that cells performed rollingcircle translation of a synthetic circular RNA.

In this Example, 0.1×10⁶ cells were plated onto each well of a 12 wellplate to monitor translation efficiency of linear or circular RNA incells. After 1 day, 1 μg of linear or circular RNA was transfected intoeach well using a lipid-based transfection reagent (Invitrogen).Twenty-four hours after transfection, cells were harvested by adding 200μl of RIPA buffer onto each well. Next, 10 μg of cell lysate proteinswere analyzed on 10-20% gradient polyacrylamide/SDS gel. Afterelectrotransfer to a nitrocellulose membrane using dry transfer method,the blot was incubated with an anti-FLAG antibody and anti-mouse IgGperoxidase. As a loading control, anti-beta tubulin antibody was used.The blot was visualized with an enhanced chemiluminescent (ECL) kit.Western blot band intensity was measured by ImageJ.

Circular RNA was successfully transfected into 293T cells, as was itslinear counterpart. However, FIG. 28 shows that 24 hours aftertransfection, EGF protein was detected in the circular RNA transfectedcells, while none was detected in the linear RNA transfected cells.Thus, circular RNA was translated in cells via rolling circletranslation as compared to linear RNA.

Example 32: Synthetic Circular RNA Demonstrated Reduced Immunogenic GeneExpression in Cells

This Example demonstrates circular RNA engineered to have reducedimmunogenicity as compared to a linear RNA.

Circular RNA that encoded a therapeutic protein provided a reducedinduction of immunogenic related genes (RIG-I, MDA5, PKA and IFN-beta)in recipient cells, as compared to linear RNA. RIG-I can recognize short5′ triphosphate uncapped double stranded or single stranded RNA, whileMDA5 can recognize longer dsRNAs. RIG-I and MDA5 can both be involved inactivating MAVS and triggering antiviral responses. PKR can be activatedby dsRNA and induced by interferons, such as IFN-beta. As shown in thefollowing Example, circular RNA was shown to have a reduced activationof an immune related genes in 293T cells than an analogous linear RNA,as assessed by expression of RIG-I, MDA5, PKR and IFN-beta by q-PCR.

The circular RNA and linear RNA were designed to encode either (1) aKozak, 3×FLAG-EGF sequence with no termination element (SEQ ID NO:11);(2) a Kozak, 3×FLAG-EGF, flanked by a termination element (stop codon)(SEQ ID NO:21); (3) a Kozak, 3×FLAG-EGF, flanked by a 2A sequence (SEQID NO:19); or (4) a Kozak, 3×FLAG-EGF sequence flanked by a 2A sequencefollowed by a termination element (stop codon) (SEQ ID NO:20).

In this Example, the level of innate immune response genes weremonitored in cells by plating 0.1×10⁶ cells into each well of a 12 wellplate. After 1 day, 1 μg of linear or circular RNA was transfected intoeach well using a lipid-based transfection reagent (Invitrogen).Twenty-four hours after transfection, total RNA was isolated from cellsusing a phenol-based extraction reagent (Invitrogen). Total RNA (500 ng)was subjected to reverse transcription to generate cDNA. qRT-PCRanalysis was performed using a dye-based quantitative PCR mix (BioRad).

Primer sequences used: Primers for GAPDH, F: AGGGCTGCTTTTAACTCTGGT (SEQID NO: 110), R: CCCCACTTGATTTTGGAGGGA (SEQ ID NO: 111); RIG-I, F:TGTGGGCAATGTCATCAAAA (SEQ ID NO: 40), R: GAAGCACTTGCTACCTCTTGC (SEQ IDNO: 41); MDA5, F: GGCACCATGGGAAGTGATT (SEQ ID NO: 42), R:ATTTGGTAAGGCCTGAGCTG (SEQ ID NO: 43); PKR, F: TCGCTGGTATCACTCGTCTG (SEQID NO: 44), R: GATTCTGAAGACCGCCAGAG (SEQ ID NO: 45); IFN-beta, F:CTCTCCTGTTGTGCTTCTCC (SEQ ID NO: 46), R: GTCAAAGTTCATCCTGTCCTTG (SEQ IDNO: 47).

As shown in FIG. 29, qRT-PCR levels of immune related genes from 293Tcells transfected with circular RNA showed reduction of RIG-I, MDA5, PKRand IFN-beta as compared to linear RNA transfected cells. Thus,induction of immunogenic related genes in recipient cells was reduced incircular RNA transfected cells, as compared to linear RNA transfectedcells.

Example 33: Increased Expression from Synthetic Circular RNA Via RollingCircle Translation in Cells

This Example demonstrates increased expression from rolling circletranslation of synthetic circular RNA in cells.

Circular RNAs were designed to include an IRES with a nanoluciferasegene or an EGF negative control gene without a termination element (stopcodon). Cells were transfected with EGF negative control (SEQ ID NO:22);nLUC stop (SEQ ID NO:23): EMCV IRES, stagger sequence (2A sequence),3×FLAG tagged nLUC sequences, stagger sequence (2A sequence), and a stopcodon; or nLUC stagger (SEQ ID NO:24): EMCV IRES, stagger sequence (2Asequence), 3×FLAG tagged nLUC sequences, and stagger sequence (2Asequence). As shown in the FIG. 30, both circular RNAs producedtranslation product having functional luciferase activity.

In this Example, translation of circular RNA was monitored in cells.Specifically, 0.1×10⁶ cells were plated onto each well of a 12 wellplate. After 1 day, 300 ng of circular RNA was transfected into eachwell using a lipid-based transfection reagent (Invitrogen). After 24hrs, cells were harvested by adding 1000 of RIPA buffer. Nanoluciferaseactivity in lysates was measured using a luciferase assay systemaccording to its manufacturer's protocol (Promega).

As shown in FIG. 30, both circular RNAs expressed protein in cells.However, circular RNA with a stagger element, e.g., 2A sequence, thatlacks a termination element (stop codon), produced higher levels ofprotein product having functional luciferase activity than circular RNAwith a termination element (stop codon).

Example 34: Synthetic Circular RNA Translated in Cells

This Example demonstrates synthetic circular RNA translation in cells.Additionally, this Example shows that circular RNA produced moreexpression product than its linear counterpart.

Circular RNA was successfully transfected into 293T cells, as was itslinear counterpart. Cells were transfected with circular RNA encodingEGF as a negative control (SEQ ID NO:22): EMCV IRES, stagger sequence(2A sequence), 3×FLAG tagged EGF sequences, stagger sequence (2Asequence); linear or circular nLUC (SEQ ID NO:23): EMCV IRES, staggersequence (2A sequence), 3×FLAG tagged nLuc sequences, a stagger sequence(2A sequence), and stop codon. As shown in FIG. 31, circular RNA wastranslated into nanoluciferase in cells.

Linear or circular RNA translation was monitored in cells. Specifically,0.1×10⁶ cells were plated onto each well of a 12 well plate. After 1day, 300 ng of linear or circular RNA was transfected into each wellusing a lipid-based transfection reagent (Invitrogen). After 24 hrs,cells were harvested by adding 100 μl of RIPA buffer. Nanoluciferaseactivity in lysates was measured using a luciferase assay systemaccording to its manufacturer's protocol (Promega).

As shown in FIG. 31, circular RNA translation product was detected incells. In particular, circular RNA had higher levels of luciferaseactivity or increased protein produced as compared to its linear RNAcounterpart.

Example 35: Rolling Circle Translation from Synthetic Circular RNAProduced Functional Protein Product in Cells

This Example demonstrates rolling circle translation of functionalprotein product from synthetic circular RNA lacking a terminationelement (stop codon), e.g., having a stagger element lacking atermination element (stop codon), in cells. Additionally, this Exampleshows that circular RNA with a stagger element expressed more functionalprotein product than its linear counterpart.

Circular RNA was successfully transfected into 293T cells, as was itslinear counterpart. Cells were transfected with circular RNA EGFnegative control (SEQ ID NO:22); linear and circular nLUC (SEQ IDNO:24): EMCV IRES, stagger sequence (2A sequence), 3×FLAG tagged nLucsequences, a stagger sequence (2A sequence). As shown in FIG. 32,circular RNA was translated into nanoluciferase in cells.

Linear or circular RNA translation was monitored in cells. Specifically,0.1×10⁶ cells were plated onto each well of a 12 well plate. After 1day, 300 ng of linear or circular RNA was transfected into each wellusing a lipid-based transfection reagent (Invitrogen). After 24 hrs,cells were harvested by adding 100 μl of RIPA buffer. Nanoluciferaseactivity in lysates was measured using a luciferase assay systemaccording to its manufacturer's protocol (Promega).

As shown in FIG. 32, circular RNA translation product was detected incells. In particular, circular RNA without a termination element (stopcodon) produced higher levels of protein product having functionalluciferase activity than its linear RNA counterpart.

Example 36: Synthetic Circular RNA Translated Via IRES Initiation inCells

This Example demonstrates synthetic circular RNA translation initiationwith an IRES in cells.

Circular RNAs were designed to include a Kozak sequence or IRES with ananoluciferase gene or an EGF negative control gene. Cells weretransfected with EGF negative control (SEQ ID NO:22), nLUC Kozak (SEQ IDNO:25): Kozak sequence, lx FLAG tagged EGF sequence, a stagger sequence(T2A sequence), 1×FLAG tagged nLUC, stagger sequence (P2A sequence), anda stop codon; or nLUC IRES (SEQ ID NO:23): EMCV IRES, stagger sequence(2A sequence), 3×FLAG tagged nLUC sequences, stagger sequence (2Asequence) and a stop codon. As shown in the FIG. 33, the circular RNAwith an IRES demonstrated higher levels of luciferase activity,corresponding to higher protein levels, as compared to circular RNA witha Kozak sequence.

In this Example, translation of circular RNA was monitored in cells.Specifically, 0.1×10⁶ cells were plated onto each well of a 12 wellplate. After 1 day, 300 ng of circular RNA was transfected into eachwell using a lipid-based transfection reagent (Invitrogen). After 24hrs, cells were harvested by adding 1000 of RIPA buffer. Nanoluciferaseactivity in lysates was measured using a luciferase assay systemaccording to its manufacturer's protocol (Promega).

As shown in FIG. 33, circular RNA initiated protein expression with anIRES and produced higher levels of protein product having functionalluciferase activity than circular RNA with Kozak initiated proteinexpression.

Example 37: Rolling Circle Translation of Synthetic Circular RNA inCells

This Example demonstrates greater protein production via rolling circletranslation of synthetic circular RNA in cells that initiated proteinproduction with an IRES.

Circular RNAs were designed to include an a Kozak sequence or IRES witha nanoluciferase gene or an EGF negative control with or without atermination element (stop codon). Cells were transfected with EGFnegative control (SEQ ID NO:22); nLUC IRES stop (SEQ ID NO:23): EMCVIRES, stagger sequence (2A sequence), 3×FLAG tagged nLUC sequences,stagger sequence (2A sequence) and a stop codon; or nLUC IRES stagger(SEQ ID NO:24): EMCV IRES, stagger sequence (2A sequence), 3×FLAG taggednLUC sequences, and stagger sequence (2A sequence). As shown in the FIG.34, both circular RNAs produced expression product demonstrated rollingcircle translation and the circular RNA without a termination element anIRES (e.g., without a Kozak sequence) initiated and produced higherlevels of protein product with functional luciferase activity thancircular RNA with a termination element out an IRES (e.g., with a Kozaksequence), demonstrating rolling circle translation.

In this Example, translation of circular RNA was monitored in cells.Specifically, 0.1×10⁶ cells were plated onto each well of a 12 wellplate. After 1 day, 300 ng of circular RNA was transfected into eachwell using a lipid-based transfection reagent (Invitrogen). After 24hrs, cells were harvested by adding 1000 of RIPA buffer. Nanoluciferaseactivity in lysates was measured using a luciferase assay systemaccording to its manufacturer's protocol (Promega).

As shown in FIG. 34, circular RNA was translated into protein in cellsvia a rolling circle method given from both circular RNAs. However, thecircular RNA that lacked a termination element (stop codon). However,the rolling circle translation of the circular RNA initiated greaterprotein production with an IRES and produced more protein product havingfunctional luciferase activity as compared to a circular RNA with atermination element Kozak translation initiation.

Example 38: Increased Protein Expressed from Circular RNA

This Example demonstrates demonstrates synthetic circular RNAtranslation in cells. Additionally, this Example shows that circular RNAproduced more expression product of the correct molecular weight thanits linear counterpart.

Linear and circular RNAs were designed to include a nanoluciferase genewith a termination element (stop codon). Cells were transfected withvehicle: transfection reagent only; linear nLUC (SEQ ID NO:23): EMCVIRES, stagger element (2A sequence), 3×FLAG tagged nLuc sequences, astagger element (2A sequence), and termination element (stop codon); orcircular nLUC (SEQ ID NO:23): EMCV IRES, stagger element (2A sequence),3×FLAG tagged nLuc sequences, a stagger element (2A sequence), and atermination element (stop codon). As shown in the FIG. 35, circular RNAproduced greater levels of protein having the correct molecular weightas compared to linear RNA.

After 24 hrs, cells were harvested by adding 100 μl of RIPA buffer.After centrifugation at 1400×g for 5 min, the supernatant was analyzedon a 10-20% gradient polyacrylamide/SDS gel.

After being electrotransferred to a nitrocellulose membrane using drytransfer method, the blot was incubated with an anti-FLAG antibody andanti-mouse IgG peroxidase. The blot was visualized with an ECL kit andwestern blot band intensity was measured by ImageJ.

As shown in FIG. 35, circular RNA was translated into protein in cells.In particular, circular RNA produced higher levels of protein having thecorrect molecular weight as compared to its linear RNA counterpart.

Example 39: Rolling Circle Translation of Synthetic Circular RNAProduced Discrete Protein Products in Cells

This Example demonstrates discrete protein products were translated viarolling circle translation from synthetic circular RNA lacking atermination element (stop codon), e.g., having a stagger element in lieuof a termination element (stop codon), in cells. Additionally, thisExample shows that circular RNA with a stagger element expressed moreprotein product having the correct molecular weight than its linearcounterpart.

Circular RNAs were designed to include a nanoluciferase gene with astagger element in place of a termination element (stop codon). Cellswere transfected with vehicle: transfection reagent only; linear nLUC(SEQ ID NO:24): EMCV IRES, stagger element (2A sequence), 3×FLAG taggednLuc sequences, and a stagger element (2A sequence); or circular nLUC(SEQ ID NO:24): EMCV IRES, stagger element (2A sequence), 3×FLAG taggednLuc sequences, and a stagger element (2A sequence). As shown in theFIG. 36, circular RNA produced greater levels of protein having thecorrect molecular weight as compared to linear RNA.

After 24 hrs, cells were harvested by adding 1000 of RIPA buffer. Aftercentrifugation at 1400×g for 5 min, the supernatant was analyzed on a10-20% gradient polyacrylamide/SDS gel.

After being electrotransferred to a nitrocellulose membrane using drytransfer method, the blot was incubated with an anti-FLAG antibody andanti-mouse IgG peroxidase. The blot was visualized with an ECL kit andwestern blot band intensity was measured by ImageJ.

As shown in FIG. 36, circular RNA translation product was detected incells. In particular, circular RNA without a termination element (stopcodon) produced higher levels of discrete protein product having thecorrect molecular weight than its linear RNA counterpart.

Example 40: Preparation of Circular RNA with a Quasi-Double Stranded,Helical Structure

This Example demonstrates circular RNA possessed both quasi-doublestranded and helical structure.

A non-naturally occurring circular RNA was engineered to adopt aquasi-double stranded, helical structure. A similar structure was shownto be involved in condensation of a naturally occurring circular RNAthat possessed a uniquely long in vivo half-life (Griffin et al 2014, JVirol. 2014 July; 88(13):7402-11. doi: 10.1128/JVI.00443-14, Guedj etal, Hepatology. 2014 December; 60(6):1902-10. doi: 10.1002/hep.27357).

In this Example, circular RNA was designed to encode a EMCV IRES, Nluctagged with 3×FLAG as ORF and stagger sequence (EMCV 2A 3×FLAG Nluc 2Ano stop). To evaluate RNA secondary structure, thermodynamic RNAstructure prediction tool (RNAfold) was used (Vienna RNA). Additionally,RNA tertiary structure was analyzed using an RNA modeling algorithm.

As shown in FIGS. 37 and 38, circular RNA is modeled to have adopted aquasi-double stranded, helical structure.

Example 41: Preparation of Circular RNA with a Quasi-Helical StructureLinked with a Repetitive Sequence

This Example demonstrates circular RNA can be designed to possess aquasi-helical structure linked with a repetitive sequence.

A non-naturally occurring circular RNA was engineered to adopt aquasi-helical structure linked with a repetitive sequence. A similarstructure was shown to be involved in condensation of a naturallyoccurring circular RNA that possessed a uniquely long in vivo half-life(Griffin et al 2014, Guedj et al 2014).

In this Example, circular RNA was designed to encode a EMCV IRES, Nlucand spacer including a repetitive sequence (SEQ ID NO: 26). To evaluateRNA tertiary structure, an RNA modeling algorithm was used.

As shown in FIG. 39, circular RNA is modeled to have adopted aquasi-helical structure.

Example 42: Circularized RNA is Circular and not Concatemeric

This Example demonstrates circular RNA degradation by RNAse H producednucleic acid degradation products consistent with a circular and not aconcatemeric RNA.

RNA, when incubated with a ligase, can either not react or form anintra- or intermolecular bond, generating a circular (no free ends) or aconcatemeric RNA, respectively. Treatment of each type of RNA with acomplementary DNA primer and RNAse H, a nonspecific endonuclease thatrecognizes DNA/RNA duplexes, is expected to produce a unique number ofdegradation products of specific sizes depending on the starting RNAmaterial.

As shown in the following Example, a ligated RNA was shown to becircular and not concatemeric based on the number and size of RNAsproduced by RNAse H degradation.

Circular RNA and linear RNA containing EMCV T2A 3×FLAG-Nluc P2A weregenerated.

To test circularization status of the RNA (1299 nts), 0.05 pmole/μl oflinear or circular RNA was incubated with 0.25 U/μl of RNAse H, anendoribonuclease that digests DNA/RNA duplexes, and 0.3 pmole/μloligomer against 1037-1046 nts of RNA (CACCGCTCAGGACAATCCTT, SEQ ID NO:55) at 37° C. for 20 min. After incubation, the reaction mixture wasanalyzed by 6% denaturing PAGE.

For the linear RNA used described above, it is expected that afterbinding of the DNA primer and subsequent cleavage by RNAse H twocleavage products are obtained of 1041 nt and 258 nt. A concatemer isexpected to produce three cleavage products of 258, 1041 and 1299 nt. Acircular is expected to produce a single 1299 nt cleavage product.

The number of bands in the linear RNA lane incubated with RNAseendonuclease produced two bands of 1041 nt and 258 nt as expected,whereas a single band of 1299nt was produced in the circular RNA lane(see FIG. 40), indicating that the circular RNA was in fact circular andnot concatemeric.

Example 43: Preparation of Large circRNAs

This Example demonstrates the generation of circular polyribonucleotidefrom in the range of about 20 bases to about 6.2 Kb.

A non-naturally occurring circular RNA engineered to include one or moredesirable properties was produced in a range of sizes depending on thedesired function. As shown in the following Example, linear RNA of up to6200 nt was circularized.

The plasmid pCDNA3.1/CAT (6.2 kb) was used here. Primers were designedto anneal to pCDNA3.1/CAT at regular intervals to generate DNAoligonucleotides corresponding to 500 nts, 1000 nts, 2000 nts, 4000 nts,5000 nts and 6200 nts. In vitro transcription of the indicated DNAoligonucleotides was performed to generate linear RNA of thecorresponding sizes. Circular RNAs were generated from these RNAoligonucleotides using splint DNA.

To measure circularization efficiency of RNA, 6 different sizes oflinear RNA (500 nts, 1000 nts, 2000 nts, 4000 nts, 5000 nts and 6200nts) were generated. They were circularized using a DNA splint and T4DNA ligase 2. As a control, one reaction was performed without T4 RNAligase. Half of the circularized sample was treated with RNAse R toremove linear RNA.

To monitor circularization efficiency, each sample was analyzed usingqPCR. As shown in FIG. 41, circular RNA was generated from a widevariety of DNA of different lengths. As shown in FIG. 42,circularization of RNA was confirmed using RNAse R treatment and qPCRanalysis against circular junctions. This Example demonstrates circularRNA production for a variety of lengths.

Example 44: Circular RNA Engineered with a Protein Binding Site

This Example demonstrates generation of a circular RNA with a proteinbinding site.

In this Example, one circular RNA is designed to include CVB3 IRES (SEQID NO:56), and an ORF encoding Gaussia luciferase (Gluc) (SEQ ID NO:57)followed by at least one protein binding site. For a specific example, aHuR binding sequence (SEQ ID NO:52) from Sindbis virus 3′UTR is used totest the effect of protein binding to circular RNA immunogenicity. HuRbinding sequence comprises two elements, URE (U-rich element; SEQ ID NO:50) and CSE (Conserved sequence element; SEQ ID NO: 51). Circular RNAwithout HuR binding sequence or with URE is used as a control. Part ofthe Anabaena autocatalytic intron and exon sequences are located priorto the CVB3 IRES (SEQ ID NO:56). The circular RNAs are generated invitro as described. As shown in FIG. 45, circular RNA was generated tocontain an HuR binding site.

To monitor the effect of RNA binding protein on circular RNAimmunogenicity, cells are plated into each well of a 96 well plate.After 1 day, 500 ng of circular RNA is transfected into each well usinga lipid-based transfection reagent (Invitrogen). Translationefficiency/RNA stability/immunogenicity are monitored daily, up to 72hrs. Media is harvested to monitor Gluc activity. Cell lysate formeasuring RNA level is prepared with a kit that allows measurements ofrelative gene expression by real-time RT-PCR (Invitrogen).

Translation efficiency is monitored by measuring Gluc activity withGaussia luciferase flash assay kit according to the manufacturer'sinstruction (Pierce).

For qRT-PCR analysis, cDNA is generated with cell lysate preparation kitaccording to manufacturer's instruction (Invitrogen). qRT-PCR analysisis performed in triplicate using a PCR master mix (Brilliant II SYBRGreen qRT-PCR Master Mix) and a PCR cycler (LightCycler 480). CircularRNA stability is measured by primers against Nluc. mRNA levels forwell-known innate immunity regulators such as RIG-I, MDAS, OAS, OASL,and PKR are quantified and normalized to actin values.

Example 45: Preparation of Circular RNA with Regulatory Nucleic AcidSites

This Example demonstrates in vitro production of circular RNA with aregulatory RNA binding site.

Different cell types possess unique nucleic acid regulatory machinery totarget specific RNA sequences. Encoding these specific sequences in acircular RNA could confer unique properties in different cell types. Asshown in the following Example, circular RNA was engineered to encode amicroRNA binding site.

In this Example, circular RNA included a sequence encoding a WT EMCVIRES, a mir692 microRNA binding site (GAGGUGCUCAAAGAGAU (SEQ ID NO:112)), and two spacer elements flanking the IRES-ORF.

The circular RNA was generated in vitro. Unmodified linear RNA was invitro transcribed from a DNA template including all the motifs listedabove, in addition to the T7 RNA polymerase promoter to drivetranscription. Transcribed RNA was purified with an RNA cleanup kit (NewEngland Biolabs, T2050), treated with RNA 5′-phosphohydrolase (RppH)(New England Biolabs, M0356) following the manufacturer's instructions,and purified again with an RNA purification column. RppH treated RNA wascircularized using a splint DNA (GGCTATTCCCAATAGCCGTT (SEQ ID NO: 113))and T4 RNA ligase 2 (New England Biolabs, M0239). Circular RNA wasUrea-PAGE purified (FIG. 43), eluted in a buffer (0.5 M Sodium Acetate,0.1% SDS, 1 mM EDTA), ethanol precipitated and resuspended in RNase freewater.

As shown in FIG. 43, circular RNA was generated with a miRNA bindingsite.

Example 46: Self-Splicing of Circular RNA

This example demonstrates the ability to produce a circular RNA byself-splicing.

For this Example, circular RNAs included a CVB3 IRES, an ORF encodingGaussia Luciferase (GLuc), and two spacer elements flanking theIRES-ORF.

The circular RNA was generated in vitro. Unmodified linear RNA was invitro transcribed from a DNA template including all the motifs listedabove. In vitro transcription reactions included 1 μg of template DNA T7RNA polymerase promoter, 10×T7 reaction buffer, 7.5 mM ATP, 7.5 mM CTP,7.5 mM GTP, 7.5 mM UTP, 10 mM DTT, 40 U RNase Inhibitor, and T7 enzyme.Transcription was carried out at 37° C. for 4h. Transcribed RNA wasDNase treated with 1 U of DNase I at 37° C. for 15 min. To favorcircularization by self splicing, additional GTP was added to a finalconcentration of 2 mM, incubated at 55° C. for 15 min. RNA was thencolumn purified and visualized by UREA-PAGE.

FIG. 44 shows circular RNA generated by self-splicing.

Example 47: Circular RNA with a Splicing Element Comprising anEncryptogen

This Example demonstrates a circular RNA engineered to have reducedimmunogenicity.

For this Example, a circular RNAs included a CVB3 IRES, an ORF encodingGaussia Luciferase (GLuc), and two spacer elements flanking theIRES-ORF, these two spacer elements comprise a splicing element that arepart of the Anabaena autocatalytic intron and exon sequences (SEQ ID NOS59 and 135).

The circular RNA is generated in vitro.

In this Example, the level of innate immune response genes is monitoredin cells by plating cells into each well of a 12 well plate. After 1day, 1 μg of linear or circular RNA is transfected into each well usinga lipid-based transfection reagent (Invitrogen). Twenty-four hours aftertransfection, total RNA is isolated from cells using a phenol-basedextraction reagent (Invitrogen). Total RNA (500 ng) is subjected toreverse transcription to generate cDNA. qRT-PCR analysis is performedusing a dye-based quantitative PCR mix (BioRad).

qRT-PCR levels of immune related genes from BJ cells transfected withcircular RNA comprising a splicing element are expected to show areduction of RIG-I, MDA5, PKR and IFN-beta as compared to linear RNAtransfected cells. Thus, induction of immunogenic related genes inrecipient cells is expected to be reduced in circular RNA transfectedcells, as compared to linear RNA transfected cells.

Example 48: Persistence of Circular RNA During Cell Division

This Example demonstrates the persistence of circular polyribonucleotideduring cell division. A non-naturally occurring circular RNA engineeredto include one or more desirable properties may persist in cells throughcell division without being degraded. As shown in the following Example,circular RNA expressing Gaussia luciferase (GLuc) was monitored over 72hdays in HeLa cells.

In this Example, a 1307nt circular RNA included a CVB3 IRES, an ORFencoding Gaussia Luciferase (GLuc), and two spacer elements flanking theIRES-ORF.

Persistence of circular RNA over cell division was monitored in HeLacells. 5000 cells/well in a 96-well plate were suspension transfectedwith circular RNA. Bright cell imaging was performed in a Avos imager(ThermoFisher) and cell counts were performed using luminescent cellviability assay (Promega) at 0 h, 24 h, 48 h, 72 h, and 96 h. GaussiaLuciferase enzyme activity was monitored daily as measure of proteinexpression and gLuc expression was monitored daily in supernatantremoved from the wells every 24 h by using the Gaussia Luciferaseactivity assay (Thermo Scientific Pierce). 50 μl of 1× Gluc substratewas added to 5 μl of plasma to carry out the Gluc luciferase activityassay. Plates were read right after mixing on a luminometer instrument(Promega).

Expression of protein from circular RNA was detected at higher levelsthan linear RNA in dividing cells (FIG. 46). Cells with circular RNA hadhigher cell division rates as compared to linear RNA at all timepointsmeasured. This Example demonstrates increased detection of circular RNAduring cell division than its linear RNA counterpart.

Example 49: Rolling Circle Translation Produced a Plurality ofExpression Sequences

This Example demonstrates the ability of circular RNA to expressmultiple proteins from a single construct. Additionally, this Exampledemonstrates rolling circle translation of circular RNA encodingmultiple ORFs. This Example further demonstrates expression of twoproteins from a single construct.

One circular RNA (mtEMCV T2A 3×FLAG-GFP F2A 3×FLAG-Nluc P2A IS spacer)was designed for rolling circle translation to include EMCV IRES (SEQ IDNO:58), and an ORF encoding GFP with 3×FLAG tag and an ORF encodingNanoluciferase (Nluc) with 3×FLAG tag. Stagger elements (2A) wereflanking the GFP and Nluc ORFs. Another circular RNA was designedsimilarly, but included a triple stop codon inbetween the Nluc ORF andthe spacer. Part of the Anabaena autocatalytic intron and exon sequenceswere included prior to the EMCV IRES. The circular RNAs were generatedeither in vitro as described.

The expression of proteins from circular RNA was monitored either invitro or in cells. For in vitro analysis, the circular RNAs wereincubated for 3h in rabbit reticulocyte lysate (Promega, Fitchburg,Wis., USA) at 30° C. The final composition of the reaction mixtureincluded 70% rabbit reticulocyte lysate, 20 μM complete amino acids, and0.8 U/μL RNase inhibitor (Toyobo, Osaka, Japan).

After incubation, hemoglobin protein was removed by adding acetic acid(0.34 μl) and water (300 μl) to the reaction mixture (16 μl) andcentrifuging at 20,817×g for 10 min at 15° C. The supernatant wasremoved and the pellet was dissolved in 2×SDS sample buffer andincubated at 70° C. for 15 min. After centrifugation at 1400×g for 5min, the supernatant was analyzed on a 10-20% gradientpolyacrylamide/SDS gel.

For analysis in cells, cells were plated into each well of a 12 wellplate to monitor translation efficiency of circular RNA in cells. After1 day, 500 ng of circular RNA was transfected into each well using alipid-based transfection reagent (Invitrogen). 48 hours aftertransfection, cells were harvested by adding 200 μl of RIPA buffer ontoeach well. Next, 10 μg of cell lysate proteins were analyzed on 10-20%gradient polyacrylamide/SDS gel.

After electrotransfer of samples from reticulocyte lysate and cells to anitrocellulose membrane using dry transfer method, the blot wasincubated with an anti-FLAG antibody and anti-mouse IgG peroxidase. As aloading control, anti-beta tubulin antibody was used. The blot wasvisualized with an enhanced chemiluminescent (ECL) kit. Western blotband intensity was measured by ImageJ.

As shown in FIG. 47, the circular RNA encoding GFP and nLuc produced 2protein products. Translation from the circular RNA without the triplestop generated more of both protein products than circular RNA with thetriple stop codon. Finally, both circular RNA with and without thetriple stop expressed proteins at 1/3.24 and 1/3.37 ratios,respectively.

Example 50: Circular RNA Shows Reduced Toxicity Compared to Linear RNA

This Example demonstrates that circular RNA is less toxic than linearRNA.

For this Example, the circular RNA includes an EMCV IRES, an ORFencoding NanoLuc with a 3×FLAG tag and flanked on either side by staggerelements (2A) and a termination element (Stop codon). The circular RNAwas generated in vitro and purified as described herein. The linear RNAused in this Example was cap-modified-poly A tailed RNA orcap-unmodified-poly A tailed RNA encoding nLuc with globin UTRs.

To monitor toxicity of RNA in cells, BJ human fibroblast cells wereplated onto each well of a 96 well plate. 50 ng of either circular orcap-modified-polyA tailed linear RNA were transfected after zero,forty-eight, and seventy-two hours, using a lipid-based transfectionreagent (ThermoFisher) following the manufacturer's recommendations.Bright cell imaging was performed in a Avos imager (ThermoFisher) at 96h. Total cells per condition were analyzed using ImageJ.

As shown in FIG. 48, transfection of circular RNA demonstrated reducedtoxicity compared to linear RNA.

Example 51: Expression Under Stress Conditions

This Example demonstrates that circular RNA expressed better understress conditions than linear RNA.

For this Example, the circular RNAs includes an EMCV IRES, an ORFencoding NanoLuc with a 3×FLAG tag, and flanked by stagger elements. Thecircular RNA was generated in vitro and purified as described. Thelinear RNA used in this Example was capped-poly A tailed RNA encodingnLuc with globin UTRs.

To monitor expression of Gaussia Luciferase from cells, BJ humanfibroblast cells were plated into each well of a 96 well plate. 50 ng ofeither circular or cap-polyA tailed linear RNA was transfected afterzero, forty-eight, and seventy-two hours, using a lipid-basedtransfection reagent following the manufacturer's recommendations.Gaussia Luciferase enzyme activity was monitored daily as measure ofprotein expression and gLuc expression was monitored daily insupernatant removed from the wells every 24h by using the GaussiaLuciferase activity assay (Thermo Scientific Pierce). 50 μl of 1× Glucsubstrate was added to 5 μl of plasma to carry out the Gluc luciferaseactivity assay. Plates were read right after mixing on a luminometerinstrument (Promega).

As shown in FIG. 49, circular RNA was translated at a higher level ascompared to linear RNA under stress condition.

Example 52: Riboswitches for Selective Expression

This Example demonstrates the ability to control protein expression fromcircular RNA.

For this Example, circular RNAs was designed to include a syntheticriboswitch (SEQ ID NO: 60) regulating the expression of the ORF encodingNanoLuc, see FIG. 50. The circular RNA was generated in vitro.Unmodified linear RNA was in vitro transcribed from a DNA templateincluding all the motifs listed above, in addition the T7 RNA polymerasepromoter to drive transcription. Transcribed RNA was purified with anRNA cleanup kit (New England Biolabs, T2050), treated with RNA5′-phosphohydrolase (RppH) (New England Biolabs, M0356) following themanufacturer's instructions, and purified again with an RNA purificationcolumn. RppH treated RNA was circularized using a splint DNA(CCGTTGTGGTCTCCCAGATAAACAGTATTTTGTCC (SEQ ID NO: 114)) and T4 RNA ligase2 (New England Biolabs, M0239). Circular RNA was Urea-PAGE purified(FIG. 50).

Theophylline or Tetracycline induce the activation of its specificriboswitch, resulting in an off-switch of gene expression (as describedby Auslander et al Mol Biosyst. 2010 May; 6(5):807-14 and Ogawa et al,RNA. 2011 March; 17(3):478-88. doi: 10.1261/rna.2433111. Epub 2011 Jan.11). It is expected that the riboswitch controls GFP or NLuc expressionfrom the circular RNA. Thus, no GFP or NLuc expression is expected afterthe addition of theophylline or tetracycline.

The efficiency of the riboswitch is tested in a cell-free translationsystem and in HeLa cells. Cell-free translation is carried out by usinga cell-free translation kit (Promega, L4140) following manufacturer'srecommendations and measuring luminescence with a luminometer instrument(Promega) for the NLuc ORF and a cell imaging multi-mode reader (BioTek)for the GFP ORF.

For cellular assays, HeLa cells/well are transfected with 1 nM of thedescribed circular RNA encoding GFP or NLuc under the control of eitherthe theophylline or the tetracycline dependent synthetic riboswitch

(first PCR forward primer for theoN5,ATACCAGCCGAAAGGCCCTTGGCAGAGAGGTCTGAAAAGACCTCTGCTGACTATGTGATCTTATTAAAATTAGG(SEQ ID NO: 115), second PCR forward primer for theoN5,GAAATTAATACGACTCACTATAGGGAGACCACAACGGTTTCCCTCCTCTATACCAGCCGAAAGGCCCTTGGCAG(SEQ ID NO: 116); first PCR forward primer for tc-N5,ACATACCAGATTTCGATCTGGAGAGGTGAAGAATACGACCACCTAGAGGTCTGAAAAGACCTCTGCTGACTATGTGATC (SEQ ID NO: 117), second PCR forward primer for tc-N5,GAAATTAATACGACTCACTATAGGGAGACCACAACGGTTTCCCTCCTCTAAAACATACCAGATTTCGATC (SEQ ID NO: 118))to assess selective expression. Lipid-based transfection reagent is usedaccording to the manufacturer's recommendations.

After 24 hr of culture at 37° C. and 5% CO2, cells are treated with andwithout theophylline or tetracycline, depending on the riboswitchencoded in the circular RNA, with concentrations ranging from 1 nM-3 mM.After 24 hrs of continuous culture, fluorescence or luminescence isevaluated. For GFP, live cells are imaged in a fluorescence neutral DMEMmedia with 5% FBS and penicillin/streptomycin and a stain for thenuclei. For NLuc, luminescence is evaluated using a luciferase system,following the manufacturer's instructions using a luminometer instrument(Promega).

(SEQ ID NO: 119)DNA template for NLuc (Blue: Plautia stali intestine virus IRES, Orange: NLuc ORF)GACACGCGGCCTTCCAAGCAGTTAGGGAAACCGACTTCTTTGAAGAAGAAAGCTGACTATGTGATCTTATTAAAATTAGGTTAAATTTCGAGGTTAAAAATAGTTTTAATATTGCTATAGTCTTAGAGGTCTTGTATATTTATACTTACCACACAAGATGGACCGGAGCAGCCCTCCAATATCTAGTGTACCCTCGTGCTCGCTCAAACATTAAGTGGTGTTGTGCGAAAAGAATCTCACTTCAAGAAAAAGAAACTAGTATGGTCTTCACACTCGAAGATTTCGTTGGGGACTGGCGACAGACAGCCGGCTACAACCTGGACCAAGTCCTTGAACAGGGAGGTGTGTCCAGTTTGTTTCAGAATCTCGGGGTGTCCGTAACTCCGATCCAAAGGATTGTCCTGAGCGGTGAAAATGGGCTGAAGATCGACATCCATGTCATCATCCCGTATGAAGGTCTGAGCGGCGACCAAATGGGCCAGATCGAAAAAATTTTTAAGGTGGTGTACCCTGTGGATGATCATCACTTTAAGGTGATCCTGCACTATGGCACACTGGTAATCGACGGGGTTACGCCGAACATGATCGACTATTTCGGACGGCCGTATGAAGGCATCGCCGTGTTCGACGGCAAAAAGATCACTGTAACAGGGACCCTGTGGAACGGCAACAAAATTATCGACGAGCGCCTGATCAACCCCGACGGCTCCCTGCTGTTCCGAGTAACCATCAACGGAGTGACCGGCTGGCGGCTGTGCGAACGCATTCTGGCGTAACTCGAGCTCGGTACCTGTCCGCGGTCGCGACGTACGCGGGCGGCCGCCATAAATTGGATCCATATATAGGGCCCGGGTTATAATTACCTCAGGTCGACGTCCCATGGTTTTGTATAGAATTTACGGCTAGCGCCGGATGCGACGCCGGTCGCGTCTTATCCGGCCTTCCTATATCAGGCGGTGTTTAAGACGCCGCCGCTTCGCCCAAATCCTTATGCCGGTTCGACGACTGGACAAAATACTG TTTATCT(SEQ ID NO: 120)DNA template for eGFP (Blue: Plautia stali intestine virus IRES, Orange: eGFP ORF)GACACGCGGCCTTCCAAGCAGTTAGGGAAACCGACTTCTTTGAAGAAGAAAGCTGACTATGTGATCTTATTAAAATTAGGTTAAATTTCGAGGTTAAAAATAGTTTTAATATTGCTATAGTCTTAGAGGTCTTGTATATTTATACTTACCACACAAGATGGACCGGAGCAGCCCTCCAATATCTAGTGTACCCTCGTGCTCGCTCAAACATTAAGTGGTGTTGTGCGAAAAGAATCTCACTTCAAGAAAAAGAAACTAGTATGGTGAGCAAGGGCGAGGAGCTGTTCACCGGGGTGGTGCCCATCCTGGTCGAGCTGGACGGCGACGTAAACGGCCACAAGTTCAGCGTGTCCGGCGAGGGCGAGGGCGATGCCACCTACGGCAAGCTGACCCTGAAGTTCATCTGCACCACCGGCAAGCTGCCCGTGCCCTGGCCCACCCTCGTGACCACCCTGACCTACGGCGTGCAGTGCTTCAGCCGCTACCCCGACCACATGAAGCAGCACGACTTCTTCAAGTCCGCCATGCCCGAAGGCTACGTCCAGGAGCGCACCATCTTCTTCAAGGACGACGGCAACTACAAGACCCGCGCCGAGGTGAAGTTCGAGGGCGACACCCTGGTGAACCGCATCGAGCTGAAGGGCATCGACTTCAAGGAGGACGGCAACATCCTGGGGCACAAGCTGGAGTACAACTACAACAGCCACAACGTCTATATCATGGCCGACAAGCAGAAGAACGGCATCAAGGTGAACTTCAAGATCCGCCACAACATCGAGGACGGCAGCGTGCAGCTCGCCGACCACTACCAGCAGAACACCCCCATCGGCGACGGCCCCGTGCTGCTGCCCGACAACCACTACCTGAGCACCCAGTCCGCCCTGAGCAAAGACCCCAACGAGAAGCGCGATCACATGGTCCTGCTGGAGTTCGTGACCGCCGCCGGGATCACTCTCGGCATGGACGAGCTGTACAAGTAACTCGAGCTCGGTACCTGTCCGCGGTCGCGACGTACGCGGGCGGCCGCCATAAATTGGATCCATATATAGGGCCCGGGTTATAATTACCTCAGGTCGACGTCCCATGGTTTTGTATAGAATTTACGGCTAGCGCCGGATGCGACGCCGGTCGCGTCTTATCCGGCCTTCCTATATCAGGCGGTGTTTAAGACGCCGCCGCTTCGCCCAAATCCTTATGCCGGTTCGACGACTGGACAAAATACTGTTTATCT (SEQ ID NO: 121) Primer SequencesForward primer for 2 (underlined: T7 promoter)GAAATTAATACGACTCACTATAGGGAGACCACAACGGTTTCCCTGACTATGTGATC(SEQ ID NO: 115)Forward primer in the 1st PCR for theoN5 (orange: aptamer; red: aIRES; purple: aaIRES)ATACCAGCCGAAAGGCCCTTGGCAGAGAGGTCTGAAAAGACCTCTGCTGACTATGTGATCTTATTAAAATTAGG (SEQ ID NO: 116) TheoN5 2nd PCRGAAATTAATACGACTCACTATAGGGAGACCACAACGGTTTCCCTCCTCTATACCAGCCGAAAGGCCCTTGGCAG (SEQ ID NO: 117) Forward primer in the 1st PCR for tc-N5ACATACCAGATTTCGATCTGGAGAGGTGAAGAATACGACCACCTAGAGGTCTGAAAAGACCTCTGCTGACTATGTGATC (SEQ ID NO: 118)Forward primer in the 2nd PCR for tc-N5GAAATTAATACGACTCACTATAGGGAGACCACAACGGTTTCCCTCCTCTAAAACATACC AGATTTCGATC(SEQ ID NO: 122) Reverse primer in all PCRsAGATAAACAGTATTTTGTCCAGTCGTCGAAC (SEQ ID NO: 123) JunctionGGACAAAATACTGTTTATCTGGGAGACCACAACGG (SEQ ID NO: 114) Splint5′-CCG TTG TGG TCT CCC AGA TAA ACA GTA TTT TGT CC-3′ 

Example 53: Circular RNA with Modified Nucleotides was Generated,Translated, and Reduced Immunogenicity of Circular RNA

This Example demonstrates the generation of modified circularpolyribonucleotide that produced protein product. In addition, thisExample demonstrates circular RNA engineered with nucleotidemodifications had reduced immunogenicity as compared to a linear RNA.

A non-naturally occurring circular RNA engineered to include one or moredesirable properties and with complete or partial incorporation ofmodified nucleotides was produced. As shown in the following Example,full length modified linear RNA or a hybrid of modified and unmodifiedlinear RNA was circularized and expression of nLuc was assessed. Inaddition, modified circular RNA was shown to have reduced activation ofimmune related genes (q-PCR of MDA5, OAS and IFN-beta expression) in BJcells, as compared to a non-modified circular RNA.

Circular RNA with a WT EMCV Nluc stop spacer was generated. For completemodification substitution, the modified nucleotides, pseudouridine andmethylcytosine or m6A, were added in place of the standard unmodifiednucleotides, uridine and cytosine or adenosine, respectively, during thein vitro transcription reaction. For the hybrid construct, the WT EMCVIRES was synthesized separately from the nLuc ORF. The WT EMCV IRES wassynthesized using either modified or non-modified nucleotides. Incontrast, the nLuc ORF sequence was synthesized using the modifiednucleotides, pseudouridine and methylcytosine or m6A, in place of thestandard unmodified nucleotides, uridine and cytosine or adenosine,respectively, during the in vitro transcription reaction. Followingsynthesis of the modified or unmodified IRES and the modified ORF, thesetwo oligonucleotides were ligated together using T4 DNA ligase. As shownin FIG. 51A modified circular RNA was generated.

To measure expression efficiency of nLuc from the fully modified orhybrid modified constructs, 0.1 pmol of linear and circular RNA wastransfected into BJ fibroblasts for 6 h. nLuc expression was measured at6 h, 24 h, 48 h and 72 h post-transfection.

The level of innate immune response genes was monitored in cells fromtotal RNA isolated from the cells using a phenol-based extractionreagent (Invitrogen). Total RNA (500 ng) was subjected to reversetranscription to generate cDNA. qRT-PCR analysis was performed using adye-based quantitative PCR mix (BioRad).

As shown in FIGS. 51B and 51C, modified circular RNA was translated. Asshown in FIGS. 52A-C, qRT-PCR levels of immune related genes from BJcells transfected with circular RNA showed reduction of MDAS, OAS andIFN-beta expression as compared to unmodified circular RNA transfectedcells. Thus, induction of immunogenic related genes in recipient cellswas reduced in cells transfected with modified circular RNA, as comparedto unmodified circular RNA transfected cells.

Example 54: Circular RNA Administrated In Vivo and Displayed a LongerHalf-Life/Increased Stability

This Example demonstrates the ability to deliver circular RNA and theincreased stability of circular RNA compared to linear RNA in vivo.

For this Example, circular RNAs were designed to include an EMCV IRESwith an ORF encoding Nanoluciferase (Nluc) and stagger sequence (EMCV 2A3×FLAG Nluc 2A no stop and EMCV 2A 3×FLAG Nluc 2A stop). The circularRNA was generated in vitro.

Balb/c mice were injected with circular RNA with Nluc ORF, or linear RNAas a control, via intravenous (IV) tail vein administration. Animalsreceived a single dose of 5 μg of RNA formulated in a lipid-basedtransfection reagent (Minis) according to manufacturer's instructions.

Mice were sacrificed, and livers were collected at 3, 4, and 7 dayspost-dosing (n=2 mice/time point). The livers were collected and storedin an RNA stabilization reagen (Invitrogen). The tissue was homogenizedin RIPA buffer with micro tube homogenizer (Fisher scientific) and RNAwas extracted using a phenol-based RNA extraction reagent for cDNAsynthesis. qPCR was used to measure the presence of both linear andcircular RNA in the liver.

RNA detection in tissues was performed by qPCR. To detect linear andcircular RNA primers that amplify the Nluc ORF were used. (F:AGATTTCGTTGGGGACTGGC (SEQ ID NO: 124), R: CACCGCTCAGGACAATCCTT (SEQ IDNO: 125)). To detect only circular RNA, primers that amplified the 5′-3′junction allowed for detection of circular but not linear RNA constructs(F: CTGGAGACGTGGAGGAGAAC (SEQ ID NO: 126), R: CCAAAAGACGGCAATATGGT (SEQID NO: 127)).

Circular RNA was detected at higher levels than linear RNA in livers ofmice at 3, 4- and 7-days post-injection (FIG. 53). Therefore, circularRNA was administered and detectable in vivo for at least 7 days postadministration.

Example 55: In Vivo Expression, Half-Life, and Non-Immunogenicity ofCircular RNA

This Example demonstrates the ability to drive expression from circularRNA in vivo. It demonstrates increased half-life of circular RNAcompared to linear RNA. Finally, it demonstrates that circular RNA wasengineered to be non-immunogenic in vivo

For this Example, circular RNAs included a CVB3 IRES, an ORF encodingGaussia Luciferase (GLuc), and two spacer elements flanking theIRES-ORF.

The circular RNA was generated in vitro. Unmodified linear RNA was invitro transcribed from a DNA template including all the motifs listedabove, as well as a T7 RNA polymerase promoter to drive transcription.Transcribed RNA was purified with an RNA cleanup kit (New EnglandBiolabs, T2050), treated with RNA 5′-phosphohydrolase (RppH) (NewEngland Biolabs, M0356) following the manufacturer's instructions, andpurified again with an RNA purification column. RppH treated RNA wascircularized using a splint DNA (GTCAACGGATTTTCCCAAGTCCGTAGCGTCTC (SEQID NO: 128)) and T4 RNA ligase 2 (New England Biolabs, M0239). CircularRNA was Urea-PAGE purified, eluted in a buffer (0.5M Sodium Acetate,0.1% SDS, 1 mM EDTA), ethanol precipitated and resuspended in RNase freewater.

Mice received a single tail vein injection dose of 2.5 μg of circularRNA with the Gaussia Luciferase ORF, or linear RNA as a control, bothformulated in a lipid-based transfection reagent (Minis) as a carrier.

Blood samples (50 μl) were collected from the tail-vein of each mouseinto EDTA tubes, at 1, 2, 7, 11, 16, and 23 days post-dosing. Plasma wasisolated by centrifugation for 25 min at 1300 g at 4° C. and theactivity of Gaussia Luciferase, a secreted enzyme, was tested using aGaussia Luciferase activity assay (Thermo Scientific Pierce). 50 μl of1× Gluc substrate was added to 5 μl of plasma to carry out the Glucluciferase activity assay. Plates were read right after mixing in aluminometer instrument (Promega).

Gaussia Luciferase activity was detected in plasma at 1, 2,7, 11, 16,and 23 days post-dosing of circular RNA (FIGS. 54A-B).

In contrast, Gaussia Luciferase activity was only detected in plasma at1, and 2 days post-dosing of modified linear RNA. Enzyme activity fromlinear RNA derived protein was not detected above background levels atday 6 or beyond (FIGS. 54A-B).

At day 16, livers were dissected from three animals and total RNA wasisolated from cells using a phenol-based extraction reagent(Invitrogen). Total RNA (500 ng) was subjected to reverse transcriptionto generate cDNA. qRT-PCR analysis was performed using a dye-basedquantitative PCR mix (BioRad).

As shown in FIG. 55, qRT-PCR levels of circular RNA but not linear RNAwere detected in both liver and spleen at day 16. As shown in FIG. 56,immune related genes from livers transfected with linear RNA showedincreased expression of RIG-I, MDAS, IFN-B and OAS, while liverstransfected with circular RNA did not show increased expression RIG-I,MDAS, PKR and IFN-beta of these markers as compared to carriertransfected animals at day 16. Thus, induction of immunogenic relatedgenes in recipient cells was not present in circular RNA fromtransfected livers.

This Example demonstrated that circular RNA expressed protein in vivofor prolonged periods of time, with levels of protein activity in theplasma at multiple days post injection. Given the half-life of GaussianLuciferase in mouse plasma is about 20 mins (see Tannous, Nat Protoc.,2009, 4(4):582-591), the similar levels of activity indicate continualexpression from circular RNA. Further, circular RNA displayed a longerexpression profile than its modified linear RNA counterpart withoutinducing immune related genes.

Sequence listing SEQ ID NO: 1 (Start Codon) AUG SEQ ID NO: 2 (GFP) EGFP:SEQ ID NO: 3  (stagger element)atggtgagcaagggcgaggagctgttcaccggggtggtgcccatcctggtcgagctggacggcgacgtaaacggccacaagttcagcgtgtccggcgagggcgagggcgatgccacctacggcaagctgaccctgaagttcatctgcaccaccggcaagctgcccgtgccctggcccaccctcgtgaccaccctgacctacggcgtgcagtgcttcagccgctaccccgaccacatgaagcagcacgacttcttcaagtccgccatgcccgaaggctacgtccaggagcgcaccatcttcttcaaggacgacggcaactacaagacccgcgccgaggtgaagttcgagggcgacaccctggtgaaccgcatcgagctgaagggcatcgacttcaaggaggacggcaacatcctggggcacaagctggagtacaactacaacagccacaacgtctatatcatggccgacaagcagaagaacggcatcaaggtgaacttcaagatccgccacaacatcgaggacggcagcgtgcagctcgccgaccactaccagcagaacacccccatcggcgacggccccgtgctgctgcccgacaaccactacctgagcacccagtccgccctgagcaaagaccccaacgagaagcgcgatcacatggtcctgctggagttcgtgaccgccgccgggatcactctcggcatggacgagctgtacaag (SEQ ID NO: 3)P2A: gctactaacttcagcctgctgaagcaggctggcgacgtggaggagaaccctggacct (SEQ ID NO: 132)T2A: gagggcaggggaagtctactaacatgcggggacgtggaggaaaatcccggccca (SEQ ID NO: 133)E2A: cagtgtactaattatgctctcttgaaattggctggagatgttgagagcaacccaggtccc Others: F2A, BmCPV2A, BmIFV2A SEQ ID NO: 4 ZKSCAN introns (SEQ ID NO: 4)GTAAAAAGAGGTGAAACCTATTATGTGTGAGCAGGGCACAGACGTTGAAACTGGAGCCAGGAGAAGTATTGGCAGGCTTTAGGTTATTAGGTGGTTACTCTGTCTTAAAAATGTTCTGGCTTTCTTCCTGCATCCACTGGCATACTCATGGTCTGTTTTTAAATATTTTAATTCCCATTTACAAAGTGATTTACCCACAAGCCCAACCTGTCTGTCTTCAG Or (SEQ ID NO: 134)GTAAGAAGCAAGGTTTCATTTAGGGGAAGGGAAATGATTCAGGACGAGAGTCTTTGTGCTGCTGAGTGCCTGTGATGAAGAAGCATGTTAGTcctgggcaacgtagcgagaccccatctctacaaaaaatagaaaaattagccaggtatagtggcgcacacctgtgattccagctacgcaggaggctgaggtgggaggattgcttgagcccaggaggttgaggctgcagtgagctgtaatcatgccactactccaacctgggcaacacagcaaggaccctgtctcaaaaGCTACTTACAGAAAAGAATTAggctcggcacggtagctcacacctgtaatcccagcactagggaggctgaggcgggcagatcacttgaggtcaggagtttgagaccagcctggccaacatggtgaaaccttgtctctactaaaaatatgaaaattagccaggcatggtggcacattcctgtaatcccagctactcgggaggctgaggcaggagaatcacttgaacccaggaggtggaggttgcagtaagccgagatcgtaccactgtgctctagccttggtgacagagcgagactgtcttaaaaaaaaaaaaaaaaaaaaaagaattaattaaaaatttaaaaaaaaatgaaaaaaaGCTGCATGCTTGTTTTTTGTTTTTAGTTATTCTACATTGTTGTCATTATTACCAAATATTGGGGAAAATACAACTTACAGACCAATCTCAGGAGTTAAATGTTACTACGAAGGCAAATGAACTATGCGTAATGAACCTGGTAGGCATTA  SEQ ID NO: 5  (IRES)IRES (EMCV):Acgttactggccgaagccgcttggaataaggccggtgtgcgtngtctatatgttattttccaccatattgccgtcttttggcaatgtgagggcccggaaacctggccctgtottcttgacgagcattcctaggggtattcccctctcgccaaaggaatgcaaggtctgttgaatgtcgtgaaggaagcagttcctctggaagottcttgaagacaaacaacgtctgtagcgaccotttgcaggcagcggaaccccccacctggcgacaggtgcctctgcggccaaaagccacgtgtataagatacacctgcaaaggcggcacaaccccagtgccacgttgtgagttggatagttgtggaaagagtcaaatggctctcctcaagcgtattcaacaaggggctgaaggatgcccagaaggtaccccattgtatgggatctgatctggggcctoggtgcacatgotttacatgtgtnagtcgaggttaaaaaacgtctaggccccccgaaccacggggacgtggttttcctttgaaaaacacgatgataata SEQ ID NO: 6  (addgene p3.1 laccase)pcDNA3.1(+) Laccase2 MCS Exon Vector sequence 6926 bpsGACGGATCGGGAGATCTCCCGATCCCCTATGGTGCACTCTCAGTACAATCTGCTCTGATGCCGCATAGTTAAGCCAGTATCTGCTCCCTGCTTGTGTGTTGGAGGTCGCTGAGTAGTGCGCGAGCAAAATTTAAGCTACAACAAGGCAAGGCTTGACCGACAATTGCATGAAGAATCTGCTTAGGGTTAGGCGTTTTGCGCTGCTTCGCGATGTACGGGCCAGATATACGCGTTGACATTGATTATTGACTAGTTATTAATAGTAATCAATTACGGGGTCATTAGTTCATAGCCCATATATGGAGTTCCGCGTTACATAACTTACGGTAAATGGCCCGCCTGGCTGACCGCCCAACGACCCCCGCCCATTGACGTCAATAATGACGTATGTTCCCATAGTAACGCCAATAGGGACTTTCCATTGACGTCAATGGGTGGAGTATTTACGGTAAACTGCCCACTTGGCAGTACATCAAGTGTATCATATGCCAAGTACGCCCCCTATTGACGTCAATGACGGTAAATGGCCCGCCTGGCATTATGCCCAGTACATGACCTTATGGGACTTTCCTACTTGGCAGTACATCTACGTATTAGTCATCGCTATTACCATGGTGATGCGGTTTTGGCAGTACATCAATGGGCGTGGATAGCGGTTTGACTCACGGGGATTTCCAAGTCTCCACCCCATTGACGTCAATGGGAGTTTGTTTTGGCACCAAAATCAACGGGACTTTCCAAAATGTCGTAACAACTCCGCCCCATTGACGCAAATGGGCGGTAGGCGTGTACGGTGGGAGGTCTATATAAGCAGAGCTCTCTGGCTAACTAGAGAACCCACTGCTTACTGGCTTATCGAAATTAATACGACTCACTATAGGGAGACCCAAGCTGGCTAGCGTTTAAACTTAAGCTTGGTACCGAGCTCGGATCCACTAGTCCAGTGTGGTGGAATTCCATTGAGAAATGACTGAGTTCCGGTGCTCTCAAGTCATTGATCTTTGTCGACTTTTATTTGGTCTCTGTAATAACGACTTCAAAAACATTAAATTCTGTTGCGAAGCCAGTAAGCTACAAAAAGAAAaaacaagagagaatgctatagtcgtatagtatagMcccgactatctgatacccattacttatctagggggaatgcgaacccaaaattttatcagttttctcggatatcgatagatattggggaataaatttaaataaataaattttgggcgggtttagggcgtggcaaaaagttttttggcaaatcgctagaaatttacaagacttataaaattatgaaaaaatacaacaaaattttaaacacgtgggcgtgacagttttggGcggttttagggcgttagagtaggcgaggacagggttacatcgactaggctttgatcctgatcaagaatatatatactttataccgcttccttctacatgttacctatttttcaacgaatctagtatacctttttactgtacgatttatgggtataaTAATAAGCTAAATCGAGACTAAGttttattgttatatatattttttttattttatGCAGAAATTAATTAAACCGGTCCTGCAGGTGATCAGGCGCGCCGGTTACCGGCCGGCCCCGCGGAGCGTAAGTATTCAAAATTCCAAAATTTTTTACTAGAAATATTCGATTTTTTAATAGGCAGTTTCTATACTATTGTATACTATTGtagattcgttgaaaagtatgtaacaggaagaataaagcatttccgaccatgtaaagtatatatattcttaataaggatcaatagccgagtcgatctcgccatgtccgtctgtcttattGattaccgccgagacatcaggaactataaaagctagaaggatgagttttagcatacagattctagagacaaggacgcagagcaagtttgttgatccatgctgccacgctttaactttctcaaattgcccaaaactgccatgcccacalttttgaactattttcgaaattttttcataattgtattactcgtgtaaatttccatcaatttgccaaaaaactttttgtcacgcgttaacgccctaaagccgccaataggtcacgcccacactattgaGcaattatcaaattttttctcattttattccccaatatctatcgatatccccgattatgaaattattaaatttcgcgttcgcattcacactagctgagtaacgagtatctgatagttggggaaatcgactTATTTTTTATATACAATGAAAATGAATTTAATCATATGAATATCGATTATAGCTTTTTATTTAATATGAATATTTATTTGGGCTTAAGGTGTAACCTcctcgacataagactcacatggcgcaggcacattgaagacaaaaatactcaTTGTCGGGTCTCGCACCCTCCAGCAGCACCTAAAATTATGTCTTCAATTATTGCCAACATTGGAGACACAATTAGTCTGTGGCACCTCAGGCGGCCGCTCGAGTCTAGAGGGCCCGTTTAAACCCGCTGATCAGCCTCGACTGTGCCTTCTAGTTGCCAGCCATCTGTTGTTTGCCCCTCCCCCGTGCCTTCCTTGACCCTGGAAGGTGCCACTCCCACTGTCCTTTCCTAATAAAATGAGGAAATTGCATCGCATTGTCTGAGTAGGTGTCATTCTATTCTGGGGGGTGGGGTGGGGCAGGACAGCAAGGGGGAGGATTGGGAAGACAATAGCAGGCATGCTGGGGATGCGGTGGGCTCTATGGCTTCTGAGGCGGAAAGAACCAGCTGGGGCTCTAGGGGGTATCCCCACGCGCCCTGTAGCGGCGCATTAAGCGCGGCGGGTGTGGTGGTTACGCGCAGCGTGACCGCTACACTTGCCAGCGCCCTAGCGCCCGCTCCTTTCGCTTTCTTCCCTTCCTTTCTCGCCACGTTCGCCGGCTTTCCCCGTCAAGCTCTAAATCGGGGGCTCCCTTTAGGGTTCCGATTTAGTGCTTTACGGCACCTCGACCCCAAAAAACTTGATTAGGGTGATGGTTCACGTAGTGGGCCATCGCCCTGATAGACGGTTTTTCGCCCTTTGACGTTGGAGTCCACGTTCTTTAATAGTGGACTCTTGTTCCAAACTGGAACAACACTCAACCCTATCTCGGTCTATTCTTTTGATTTATAAGGGATTTTGCCGATTTCGGCCTATTGGTTAAAAAATGAGCTGATTTAACAAAAATTTAACGCGAATTAATTCTGTGGAATGTGTGTCAGTTAGGGTGTGGAAAGTCCCCAGGCTCCCCAGCAGGCAGAAGTATGCAAAGCATGCATCTCAATTAGTCAGCAACCAGGTGTGGAAAGTCCCCAGGCTCCCCAGCAGGCAGAAGTATGCAAAGCATGCATCTCAATTAGTCAGCAACCATAGTCCCGCCCCTAACTCCGCCCATCCCGCCCCTAACTCCGCCCAGTTCCGCCCATTCTCCGCCCCATGGCTGACTAATTTTTTTTATTTATGCAGAGGCCGAGGCCGCCTCTGCCTCTGAGCTATTCCAGAAGTAGTGAGGAGGCTTTTTTGGAGGCCTAGGCTTTTGCAAAAAGCTCCCGGGAGCTTGTATATCCATTTTCGGATCTGATCAAGAGACAGGATGAGGATCGTTTCGCATGATTGAACAAGATGGATTGCACGCAGGTTCTCCGGCCGCTTGGGTGGAGAGGCTATTCGGCTATGACTGGGCACAACAGACAATCGGCTGCTCTGATGCCGCCGTGTTCCGGCTGTCAGCGCAGGGGCGCCCGGTTCTTTTTGTCAAGACCGACCTGTCCGGTGCCCTGAATGAACTGCAGGACGAGGCAGCGCGGCTATCGTGGCTGGCCACGACGGGCGTTCCTTGCGCAGCTGTGCTCGACGTTGTCACTGAAGCGGGAAGGGACTGGCTGCTATTGGGCGAAGTGCCGGGGCAGGATCTCCTGTCATCTCACCTTGCTCCTGCCGAGAAAGTATCCATCATGGCTGATGCAATGCGGCGGCTGCATACGCTTGATCCGGCTACCTGCCCATTCGACCACCAAGCGAAACATCGCATCGAGCGAGCACGTACTCGGATGGAAGCCGGTCTTGTCGATCAGGATGATCTGGACGAAGAGCATCAGGGGCTCGCGCCAGCCGAACTGTTCGCCAGGCTCAAGGCGCGCATGCCCGACGGCGAGGATCTCGTCGTGACCCATGGCGATGCCTGCTTGCCGAATATCATGGTGGAAAATGGCCGCTTTTCTGGATTCATCGACTGTGGCCGGCTGGGTGTGGCGGACCGCTATCAGGACATAGCGTTGGCTACCCGTGATATTGCTGAAGAGCTTGGCGGCGAATGGGCTGACCGCTTCCTCGTGCTTTACGGTATCGCCGCTCCCGATTCGCAGCGCATCGCCTTCTATCGCCTTCTTGACGAGTTCTTCTGAGCGGGACTCTGGGGTTCGAAATGACCGACCAAGCGACGCCCAACCTGCCATCACGAGATTTCGATTCCACCGCCGCCTTCTATGAAAGGTTGGGCTTCGGAATCGTTTTCCGGGACGCCGGCTGGATGATCCTCCAGCGCGGGGATCTCATGCTGGAGTTCTTCGCCCACCCCAACTTGTTTATTGCAGCTTATAATGGTTACAAATAAAGCAATAGCATCACAAATTTCACAAATAAAGCATTTTYTTCACTGCATTCTAGTTGTGGTTTGTCCAAACTCATCAATGTATCTTATCATGTCTGTATACCGTCGACCTCTAGCTAGAGCTTGGCGTAATCATGGTCATAGCTGTTTCCTGTGTGAAATTGTTATCCGCTCACAATTCCACACAACATACGAGCCGGAAGCATAAAGTGTAAAGCCTGGGGTGCCTAATGAGTGAGCTAACTCACATTAATTGCGTTGCGCTCACTGCCCGCTTTCCAGTCGGGAAACCTGTCGTGCCAGCTGCATTAATGAATCGGCCAACGCGCGGGGAGAGGCGGTTTGCGTATTGGGCGCTCTTCCGCTTCCTCGCTCACTGACTCGCTGCGCTCGGTCGTTCGGCTGCGGCGAGCGGTATCAGCTCACTCAAAGGCGGTAATACGGTTATCCACAGAATCAGGGGATAACGCAGGAAAGAACATGTGAGCAAAAGGCCAGCAAAAGGCCAGGAACCGTAAAAAGGCCGCGTTGCTGGCGTTTTTCCATAGGCTCCGCCCCCCTGACGAGCATCACAAAAATCGACGCTCAAGTCAGAGGTGGCGAAACCCGACAGGACTATAAAGATACCAGGCGTTTCCCCCTGGAAGCTCCCTCGTGCGCTCTCCTGTTCCGACCCTGCCGCTTACCGGATACCTGTCCGCCTTTCTCCCTTCGGGAAGCGTGGCGCTTTCTCATAGCTCACGCTGTAGGTATCTCAGTTCGGTGTAGGTCGTTCGCTCCAAGCTGGGCTGTGTGCACGAACCCCCCGTTCAGCCCGACCGCTGCGCCTTATCCGGTAACTATCGTCTTGAGTCCAACCCGGTAAGACACGACTTATCGCCACTGGCAGCAGCCACTGGTAACAGGATTAGCAGAGCGAGGTATGTAGGCGGTGCTACAGAGTTCTTGAAGTGGTGGCCTAACTACGGCTACACTAGAAGAACAGTATTTGGTATCTGCGCTCTGCTGAAGCCAGTTACCTTCGGAAAAAGAGTTGGTAGCTCTTGATCCGGCAAACAAACCACCGCTGGTAGCGGTTTTTTTGTTTGCAAGCAGCAGATTACGCGCAGAAAAAAAGGATCTCAAGAAGATCCTTTGATCTTTTCTACGGGGTCTGACGCTCAGTGGAACGAAAACTCACGTTAAGGGATTTTGGTCATGAGATTATCAAAAAGGATCTTCACCTAGATCCTTTTAAATTAAAAATGAAGTTTTAAATCAATCTAAAGTATATATGAGTAAACTTGGTCTGACAGTTACCAATGCTTAATCAGTGAGGCACCTATCTCAGCGATCTGTCTATTTCGTTCATCCATAGTTGCCTGACTCCCCGTCGTGTAGATAACTACGATACGGGAGGGCTTACCATCTGGCCCCAGTGCTGCAATGATACCGCGAGACCCACGCTCACCGGCTCCAGATTTATCAGCAATAAACCAGCCAGCCGGAAGGGCCGAGCGCAGAAGTGGTCCTGCAACTTTATCCGCCTCCATCCAGTCTATTAATTGTTGCCGGGAAGCTAGAGTAAGTAGTTCGCCAGTTAATAGTTTGCGCAACGTTGTTGCCATTGCTACAGGCATCGTGGTGTCACGCTCGTCGTTTGGTATGGCTTCATTCAGCTCCGGTTCCCAACGATCAAGGCGAGTTACATGATCCCCCATGTTGTGCAAAAAAGCGGTTAGCTCCTTCGGTCCTCCGATCGTTGTCAGAAGTAAGTTGGCCGCAGTGTTATCACTCATGGTTATGGCAGCACTGCATAATTCTCTTACTGTCATGCCATCCGTAAGATGCTTTTCTGTGACTGGTGAGTACTCAACCAAGTCATTCTGAGAATAGTGTATGCGGCGACCGAGTTGCTCTTGCCCGGCGTCAATACGGGATAATACCGCGCCACATAGCAGAACTTTAAAAGTGCTCATCATTGGAAAACGTTCTTCGGGGCGAAAACTCTCAAGGATCTTACCGCTGTTGAGATCCAGTTCGATGTAACCCACTCGTGCACCCAACTGATCTTCAGCATCTTTTACTTTCACCAGCGTTTCTGGGTGAGCAAAAACAGGAAGGCAAAATGCCGCAAAAAAGGGAATAAGGGCGACACGGAAATGTTGAATACTCATACTCTTCCTTTTTCAATATTATTGAAGCATTTATCAGGGTTATTGTCTCATGAGCGGATACATATTTGAATGTATTTAGAAAAATAAACAAATAGGGGTTCCGCGCACATTTCCCCGAAAAGTGCCACCTGAC GTCSEQ ID NO: 8 (RFP) mCherry:atggtgagcaagggcgaggaggataacatggccatcatcaaggagttcatgcgcttcaaggtgcacatggagggctccgtgaacggccacgagttcgagatcgagggcgagggcgagggccgcccctacgagggcacccagaccgccaagctgaaggtgaccaagggtggccccctgcccttcgcctgggacatcctgtcccctcagttcatgtacggctccaaggcctacgtgaagcaccccgccgacatccccgactacttgaagctgtccttccccgagggcttcaagtgggagcgcgtgatgaacttcgaggacggcggcgtggtgaccgtgacccaggactcctccctgcaggacggcgagttcatctacaaggtgaagctgcgcggcaccaacttcccctccgacggccccgtaatgcagaagaagaccatgggctgggaggcctcctccgagcggatgtaccccgaggacggcgccctgaagggcgagatcaagcagaggctgaagctgaaggacggcggccactacgacgctgaggtcaagaccacctacaaggccaagaagcccgtgcagctgcccggcgcctacaacgtcaacatcaagttggacatcacctcccacaacgaggactacaccatcgtggaacagtacgaacgcgccgagggccgccactccaccggcggcatggacgagctgtacaag SEQ ID NO: 9  (riboswitch)Aptazyme (Theophylline Dependent see Auslander 2010 Mol Biosys):cugagaugcagguacauccagcugaugagucccaaauaggacgaaagccauaccagccgaaaggcccuuggcaggguuccuggauuccacugcuauccac SEQ ID NO: 10  (luciferase) nLuc:ATGGTCTTCACACTCGAAGATTTCGTTGGGGACTGGCGACAGACAGCCGGCTACAACCTGGACCAAGTCCTTGAACAGGGAGGTGTGTCCAGTTTGTTTCAGAATCTCGGGGTGTCCGTAACTCCGATCCAAAGGATTGTCCTGAGCGGTGAAAATGGGCTGAAGATCGACATCCATGTCATCATCCCGTATGAAGGTCTGAGCGGCGACCAAATGGGCCAGATCGAAAAAATTTTTAAGGTGGTGTACCCTGTGGATGATCATCACTTTAAGGTGATCCTGCACTATGGCACACTGGTAATCGACGGGGTTACGCCGAACATGATCGACTATTTCGGACGGCCGTATGAAGGCATCGCCGTGTTCGACGGCAAAAAGATCACTGTAACAGGGACCCTGTGGAACGGCAACAAAATTATCGACGAGCGCCTGATCAACCCCGACGGCTCCCTGCTGTTCCGAGTAACCATCAACGGAGTGACCGGCTGGCGGCTGTGCGAACGCATTCTG GCGTAASequence ID 11 Kozak3XFLAG-EGF nostop (264 bps)GGGAGCCACCATGGACTACAAGGACGACGACGACAAGATCATCGACTATAAAGACGACGACGATAAAGGTGGCGACTATAAGGACGACGACGACAAAGCCATTAATAGTGACTCTGAGTGTCCCCTGTCCCACGACGGGTACTGCCTCCACGACGGTGTGTGCATGTATATTGAAGCATTGGACAAGTACGCCTGCAACTGTGTTGTTGGCTACATCGGGGAGCGCTGTCAGTACCGAGACCTGAAGTGGTGGGAACTGCGCCT  5-13: Kozak sequence 14-262:3XFLAG-EGF SEQ ID NO: 12Kozak 3XFLAG-EGF stop (273 bps)GGGAGCCACCATGGACTACAAGGACGACGACGACAAGATCATCGACTATAAAGACGACGACGATAAAGGTGGCGACTATAAGGACGACGACGACAAAGCCATTAATAGTGACTCTGAGTGTCCCCTGTCCCACGACGGGTACTGCCTCCACGACGGTGTGTGCATGTATATTGAAGCATTGGACAAGTACGCCTGCAACTGTGTTGTTGGCTACATCGGGGAGCGCTGTCAGTACCGAGACCTGAAGTGGTGGGAACTGCGCTGATAGTAACT   5-13: Kozak sequence  14-262: 3XFLAG-EGF263-271: Triple stop codon SEQ ID NO: 13Kozak 3XFLAG-EGF P2A nostop (330 bps)GGGAGCCACCATGGACTACAAGGACGACGACGACAAGATCATCGACTATAAAGACGACGACGATAAAGGTGGCGACTATAAGGACGACGACGACAAAGCCATTAATAGTGACTCTGAGTGTCCCCTGTCCCACGACGGGTACTGCCTCCACGACGGTGTGTGCATGTATATTGAAGCATTGGACAAGTACGCCTGCAACTGTGTTGTTGGCTACATCGGGGAGCGCTGTCAGTACCGAGACCTGAAGTGGTGGGAACTGCGCGGAAGCGGAGCTACTAACTTCAGCCTGCTGAAGCAGGCTGGAGACGTGGAGGAGAACCCTGGACCTCT   5-13: Kozak sequence  14-262: 3XFLAG-EGF 263-328: P2ASEQ ID NO: 14 Splint for construct Kozak 3XFLAG-EGF nostop (264 bps)GGTGGCTCCCAGGCGCAGTT SEQ ID NO: 15Splint for construct Kozak 3XFLAG-EGF stop (273 bps)GCTTGGCTCCCAGTTACTATC SEQ ID NO: 16Splint for construct Kozak 3XFLAG-EGF P2A nostop (330 bps)GGTGGCTCCCAGAGGTCCAG SEQ ID NO: 17Kozak 1XFLAG-EGF T2A 1XFLAG-Nluc P2A nostop (873 bps)GGGAGCCACCATGGACTACAAGGACGACGACGACAAGATCATCAATAGTGACTCTGAGTGTCCCCTGTCCCACGACGGGTACTGCCTCCACGACGGTGTGTGCATGTATATTGAAGCATTGGACAAGTACGCCTGCAACTGTGTTGTTGGCTACATCGGGGAGCGCTGTCAGTACCGAGACCTGAAGTGGTGGGAACTGCGCGGCTCCGGCGAGGGCAGGGGAAGTCTTCTAACATGCGGGGACGTGGAGGAAAATCCCGGCCCAGACTATAAGGACGACGACGACAAAATCATCGTCTTCACACTCGAAGATTTCGTTGGGGACTGGCGACAGACAGCCGGCTACAACCTGGACCAAGTCCTTGAACAGGGAGGTGTGTCCAGTTTGTTTCAGAATCTCGGGGTGTCCGTAACTCCGATCCAAAGGATTGTCCTGAGCGGTGAAAATGGGCTGAAGATCGACATCCATGTCATCATCCCGTATGAAGGTCTGAGCGGCGACCAAATGGGCCAGATCGAAAAAATTTTTAAGGTGGTGTACCCTGTGGATGATCATCACTTTAAGGTGATCCTGCACTATGGCACACTGGTAATCGACGGGGTTACGCCGAACATGATCGACTATTTCGGACGGCCGTATGAAGGCATCGCCGTGTTCGACGGCAAAAAGATCACTGTAACAGGGACCCTGTGGAACGGCAACAAAATTATCGACGAGCGCCTGATCAACCCCGACGGCTCCCTGCTGTTCCGAGTAACCATCAACGGAGTGACCGGCTGGCGGCTGTGCGAACGCATTCTGGCGGGAAGCGGAGCTACTAACTTCAGCCTGCTGAAGCAGGCTGGAGACGTGGAGGAGAACCCTGGACCTCT   5-13: Kozak sequence 14-202: 1XFLAG-EGF 203-265: T2A 266-805: 1XFLAG-Nluc 806-871: P2ASEQ ID NO: 18 Kozak 1XFLAG-EGF stop 1XFLAG-Nluc stop (762 bps)GGGAGCCACCATGGACTACAAGGACGACGACGACAAGATCATCAATAGTGACTCTGAGTGTCCCCTGTCCCACGACGGGTACTGCCTCCACGACGGTGTGTGCATGTATATTGAAGCATTGGACAAGTACGCCTGCAACTGTGTTGTTGGCTACATCGGGGAGCGCTGTCAGTACCGAGACCTGAAGTGGTGGGAACTGCGCTGATAGTAAGACTATAAGGACGACGACGACAAAATCATCGTCTTCACACTCGAAGATTTCGTTGGGGACTGGCGACAGACAGCCGGCTACAACCTGGACCAAGTCCTTGAACAGGGAGGTGTGTCCAGTTTGTTTCAGAATCTCGGGGTGTCCGTAACTCCGATCCAAAGGATTGTCCTGAGCGGTGAAAATGGGCTGAAGATCGACATCCATGTCATCATCCCGTATGAAGGTCTGAGCGGCGACCAAATGGGCCAGATCGAAAAAATTTTTAAGGTGGTGTACCCTGTGGATGATCATCACTTTAAGGTGATCCTGCACTATGGCACACTGGTAATCGACGGGGTTACGCCGAACATGATCGACTATTTCGGACGGCCGTATGAAGGCATCGCCGTGTTCGACGGCAAAAAGATCACTGTAACAGGGACCCTGTGGAACGGCAACAAAATTATCGACGAGCGCCTGATCAACCCCGACGGCTCCCTGCTGTTCCGAGTAACCATCAACGGAGTGACCGGCTGGCGGCTGTGCGAACGCATTCTGGCGTGATAGTAACT  5-13: Kozak sequence  14-202: 1XFLAG-EGF 203-211: Triple stop codon212-751: 1XFLAG-Nluc 752-760: Triple stop codon SEQ ID NO: 19Kozak 3XFLAG-EGF P2A nostop (330 bps)GGGAGCCACCATGGACTACAAGGACGACGACGACAAGATCATCGACTATAAAGACGACGACGATAAAGGTGGCGACTATAAGGACGACGACGACAAAGCCATTAATAGTGACTCTGAGTGTCCCCTGTCCCACGACGGGTACTGCCTCCACGACGGTGTGTGCATGTATATTGAAGCATTGGACAAGTACGCCTGCAACTGTGTTGTTGGCTACATCGGGGAGCGCTGTCAGTACCGAGACCTGAAGTGGTGGGAACTGCGCGGAAGCGGAGCTACTAACTTCAGCCTGCTGAAGCAGGCTGGAGACGTGGAGGAGAACCCTGGACCTCT   5-13: Kozak sequence  14-262: 3XFLAG-EGF 263-328: P2ASEQ ID NO: 20 Kozak 3XFLAG-EGF nostop (264 bps)GGGAGCCACCATGGACTACAAGGACGACGACGACAAGATCATCGACTATAAAGACGACGACGATAAAGGTGGCGACTATAAGGACGACGACGACAAAGCCATTAATAGTGACTCTGAGTGTCCCCTGTCCCACGACGGGTACTGCCTCCACGACGGTGTGTGCATGTATATTGAAGCATTGGACAAGTACGCCTGCAACTGTGTTGTTGGCTACATCGGGGAGCGCTGTCAGTACCGAGACCTGAAGTGGTGGGAACTGCGCCT  5-13: Kozak sequence 14-262: 3XFLAG-EGF SEQ ID NO: 21Kozak 3XFLAG-EGFstop (273 bps)GGGAGCCACCATGGACTACAAGGACGACGACGACAAGATCATCGACTATAAAGACGACGACGATAAAGGTGGCGACTATAAGGACGACGACGACAAAGCCATTAATAGTGACTCTGAGTGTCCCCTGTCCCACGACGGGTACTGCCTCCACGACGGTGTGTGCATGTATATTGAAGCATTGGACAAGTACGCCTGCAACTGTGTTGTTGGCTACATCGGGGAGCGCTGTCAGTACCGAGACCTGAAGTGGTGGGAACTGCGCTGATAGTAACT   5-13: Kozak sequence  14-262: 3XFLAG-EGF263-271: Triple stop codon SEQ ID NO: 22EMCV IRES T2A 3XFLAG-EGF P2A nostop (954 bps)GGGACCTAACGTTACTGGCCGAAGCCGCTTGGAACAAGGCCGGTGTGCGTTTGTCTATATGTTATTTTCCACCATATTGCCGTCTTTTGGCAATGTGAGGGCCCGGAAACCTGGCCCTGTCTTCTTGACGAGCATTCCTAGGGGTCTTTCCCCTCTCGCCAAAGGAATGCAAGGTCTGTTGAATGTCGTGAAGGAAGCAGTTCCTCTGGAAGCTTCTTCAAGACAAACAACGTCTGTAGCGACCCTTTGCAGGCAGCGGAACCCCCCACCTGGCGACAGGTGCCTCTGCGGCCAAAAGCCACGTGTATACGATACACCTGCAAAGGCGGCACAACCCCAGTGCCACGTTGTGAGTTGGATAGTTGTGGAAAGAGTCAAATGGCTCTCCTCAAGCGTATTCAACAAGGGGCTGAAGGATGCCCAGAAGGTACCCCATTGTATGGGATCTGATCTGGGGCCTCGGTGCACATGCTTTACATGTGTTCAGTCGAGGTTAAAAAACGTCCAGGCCCCCCGAACCACGGGGACGTGGTTTTCCTTTGAAAAACACGATGATAATATGGCCACAACCATGGGCTCCGGCGAGGGCAGGGGAAGTCTTCTAACATGCGGGGACGTGGAGGAAAATCCCGGCCCAGACTACAAGGACGACGACGACAAGATCATCGACTATAAAGACGACGACGATAAAGGTGGCGACTATAAGGACGACGACGACAAAGCCATTAATAGTGACTCTGAGTGTCCCCTGTCCCACGACGGGTACTGCCTCCACGACGGTGTGTGCATGTATATTGAAGCATTGGACAAGTACGCCTGCAACTGTGTTGTTGGCTACATCGGGGAGCGCTGTCAGTACCGAGACCTGAAGTGGTGGGAACTGCGCGGAAGCGGAGCTACTAACTTCAGCCTGCTGAAGCAGGCTGGAGACGTGGAGGAGAACCCTGGACCTCT  5-574: EMCV IRES 575-637: T2A 638-886: 3XFALG-EGF 887-952: P2ASEQ ID NO: 23 EMCV T2A 3XFLAG Nluc P2A stop (1314 nts)GGGACCTAACGTTACTGGCCGAAGCCGCTTGGAACAAGGCCGGTGTGCGTTTGTCTATATGTTATTTTCCACCATATTGCCGTCTTTTGGCAATGTGAGGGCCCGGAAACCTGGCCCTGTCTTCTTGACGAGCATTCCTAGGGGTCTTTCCCCTCTCGCCAAAGGAATGCAAGGTCTGTTGAATGTCGTGAAGGAAGCAGTTCCTCTGGAAGCTTCTTCAAGACAAACAACGTCTGTAGCGACCCTTTGCAGGCAGCGGAACCCCCCACCTGGCGACAGGTGCCTCTGCGGCCAAAAGCCACGTGTATACGATACACCTGCAAAGGCGGCACAACCCCAGTGCCACGTTGTGAGTTGGATAGTTGTGGAAAGAGTCAAATGGCTCTCCTCAAGCGTATTCAACAAGGGGCTGAAGGATGCCCAGAAGGTACCCCATTGTATGGGATCTGATCTGGGGCCTCGGTGCACATGCTTTACATGTGTTCAGTCGAGGTTAAAAAACGTCCAGGCCCCCCGAACCACGGGGACGTGGTTTTCCTTTGAAAAACACGATGATAATATGGCCACAACCATGGGCTCCGGCGAGGGCAGGGGAAGTCTTCTAACATGCGGGGACGTGGAGGAAAATCCCGGCCCAGACTACAAGGACGACGACGACAAGATCATCGACTATAAAGACGACGACGATAAAGGTGGCGACTATAAGGACGACGACGACAAAGCCATTGTCTTCACACTCGAAGATTTCGTTGGGGACTGGCGACAGACAGCCGGCTACAACCTGGACCAAGTCCTTGAACAGGGAGGTGTGTCCAGTTTGTTTCAGAATCTCGGGGTGTCCGTAACTCCGATCCAAAGGATTGTCCTGAGCGGTGAAAATGGGCTGAAGATCGACATCCATGTCATCATCCCGTATGAAGGTCTGAGCGGCGACCAAATGGGCCAGATCGAAAAAATTTTTAAGGTGGTGTACCCTGTGGATGATCATCACTTTAAGGTGATCCTGCACTATGGCACACTGGTAATCGACGGGGTTACGCCGAACATGATCGACTATTTCGGACGGCCGTATGAAGGCATCGCCGTGTTCGACGGCAAAAAGATCACTGTAACAGGGACCCTGTGGAACGGCAACAAAATTATCGACGAGCGCCTGATCAACCCCGACGGCTCCCTGCTGTTCCGAGTAACCATCAACGGAGTGACCGGCTGGCGGCTGTGCGAACGCATTCTGGCGGGAAGCGGAGCTACTAACTTCAGCCTGCTGAAGCAGGCTGGAGACGTGGAGGAGAACCCTGGACCTTGATAGTAACT    5-574: EMCV IRES  575-637: T2A 638-1237: 3XFLAG Nluc 1238-1303: P2A 1304-1312: Triple stop codonSEQ ID NO: 24 EMCV T2A 3XFLAG Nluc P2A nostop (1305 nts)GGGACCTAACGTTACTGGCCGAAGCCGCTTGGAACAAGGCCGGTGTGCGTTTGTCTATATGTTATTTTCCACCATATTGCCGTCTTTTGGCAATGTGAGGGCCCGGAAACCTGGCCCTGTCTTCTTGACGAGCATTCCTAGGGGTCTTTCCCCTCTCGCCAAAGGAATGCAAGGTCTGTTGAATGTCGTGAAGGAAGCAGTTCCTCTGGAAGCTTCTTCAAGACAAACAACGTCTGTAGCGACCCTTTGCAGGCAGCGGAACCCCCCACCTGGCGACAGGTGCCTCTGCGGCCAAAAGCCACGTGTATACGATACACCTGCAAAGGCGGCACAACCCCAGTGCCACGTTGTGAGTTGGATAGTTGTGGAAAGAGTCAAATGGCTCTCCTCAAGCGTATTCAACAAGGGGCTGAAGGATGCCCAGAAGGTACCCCATTGTATGGGATCTGATCTGGGGCCTCGGTGCACATGCTTTACATGTGTTCAGTCGAGGTTAAAAAACGTCCAGGCCCCCCGAACCACGGGGACGTGGTTTTCCTTTGAAAAACACGATGATAATATGGCCACAACCATGGGCTCCGGCGAGGGCAGGGGAAGTCTTCTAACATGCGGGGACGTGGAGGAAAATCCCGGCCCAGACTACAAGGACGACGACGACAAGATCATCGACTATAAAGACGACGACGATAAAGGTGGCGACTATAAGGACGACGACGACAAAGCCATTGTCTTCACACTCGAAGATTTCGTTGGGGACTGGCGACAGACAGCCGGCTACAACCTGGACCAAGTCCTTGAACAGGGAGGTGTGTCCAGTTTGTTTCAGAATCTCGGGGTGTCCGTAACTCCGATCCAAAGGATTGTCCTGAGCGGTGAAAATGGGCTGAAGATCGACATCCATGTCATCATCCCGTATGAAGGTCTGAGCGGCGACCAAATGGGCCAGATCGAAAAAATTTTTAAGGTGGTGTACCCTGTGGATGATCATCACTTTAAGGTGATCCTGCACTATGGCACACTGGTAATCGACGGGGTTACGCCGAACATGATCGACTATTTCGGACGGCCGTATGAAGGCATCGCCGTGTTCGACGGCAAAAAGATCACTGTAACAGGGACCCTGTGGAACGGCAACAAAATTATCGACGAGCGCCTGATCAACCCCGACGGCTCCCTGCTGTTCCGAGTAACCATCAACGGAGTGACCGGCTGGCGGCTGTGCGAACGCATTCTGGCGGGAAGCGGAGCTACTAACTTCAGCCTGCTGAAGCAGGCTGGAGACGTGGAGGAGAACCCTGGACCTCT    5-574: EMCV IRES  575-637: T2A 638-1237: 3XFLAG Nluc 1238-1303: P2A SEQ ID NO: 25Kozak 1XFLAG-EGF T2A 1XFLAG-NLuc P2A stop (882 bps)GGGAGCCACCATGGACTACAAGGACGACGACGACAAGATCATCAATAGTGACTCTGAGTGTCCCCTGTCCCACGACGGGTACTGCCTCCACGACGGTGTGTGCATGTATATTGAAGCATTGGACAAGTACGCCTGCAACTGTGTTGTTGGCTACATCGGGGAGCGCTGTCAGTACCGAGACCTGAAGTGGTGGGAACTGCGCGGCTCCGGCGAGGGCAGGGGAAGTCTTCTAACATGCGGGGACGTGGAGGAAAATCCCGGCCCAGACTATAAGGACGACGACGACAAAATCATCGTCTTCACACTCGAAGATTTCGTTGGGGACTGGCGACAGACAGCCGGCTACAACCTGGACCAAGTCCTTGAACAGGGAGGTGTGTCCAGTTTGTTTCAGAATCTCGGGGTGTCCGTAACTCCGATCCAAAGGATTGTCCTGAGCGGTGAAAATGGGCTGAAGATCGACATCCATGTCATCATCCCGTATGAAGGTCTGAGCGGCGACCAAATGGGCCAGATCGAAAAAATTTTTAAGGTGGTGTACCCTGTGGATGATCATCACTTTAAGGTGATCCTGCACTATGGCACACTGGTAATCGACGGGGTTACGCCGAACATGATCGACTATTTCGGACGGCCGTATGAAGGCATCGCCGTGTTCGACGGCAAAAAGATCACTGTAACAGGGACCCTGTGGAACGGCAACAAAATTATCGACGAGCGCCTGATCAACCCCGACGGCTCCCTGCTGTTCCGAGTAACCATCAACGGAGTGACCGGCTGGCGGCTGTGCGAACGCATTCTGGCGGGAAGCGGAGCTACTAACTTCAGCCTGCTGAAGCAGGCTGGAGACGTGGAGGAGAACCCTGGACCTTGATAGTAACT  5-13: Kozak sequence  14-202: 1XFLAG-EGF 266-805: 1XFLAG-NLuc806-871: P2A 872-880: Triple stop codon SEQ ID NO: 26Exemplary Repetitive Spacer SequenceAAAAAACAAAAAACAAAACGGCTATTATGCGTTACCGGCGAGACGCTACGGACTTGGGAAAATCCGTTGACCTTAAACGGTCGTGTGGGTTCAAGTCCCTCCACCCCCACGCCGGAAACGCAATAGCCGAAAAACAAAAAACAAAAAAAACAAAAAAAAAACCAAAAAAACAAAACACA SEQ ID NO: 27Forward primer used in Example 43 to amplify template from pCDNA3.1/CATCGCGGATCCTAATACGACTCACTATAGGGAGACCCAAGCTGGC SEQ ID NO: 28Reverse primer used in Example 43 to amplify 0.5 kb template from pCDNA3.1/CATAATAGCCGTTTTGTTTTTTGGATTACCAGTGTGCCATAGTGCAGGATCACATCGTCGTGGTATTCACTCCAGAGCGATG SEQ ID NO: 29Reverse primer used in Example 43 to amplify l kb template from pCDNA3.1/CATAATAGCCGTTTTGTTTTTTGGATTACCAGTGTGCCATAGTGCAGGATCACACGGGGGAGGGGCAAACAACAGATGG SEQ ID NO: 30Reverse primer used in Example 43 to amplify 2 kb template from pCDNA3.1/CATAATAGCCGTTTTGTTTTTTGGATTACCAGTGTGCCATAGTGCAGGATCACGCTTTTTGCAAAAGCCTAGGCCTCCAAAAAAGCC SEQ ID NO: 31Reverse primer used in Example 43 to amplify 4 kb template from pCDNA3.1/CATAATAGCCGTTTTGTTTTTTGGATTACCAGTGTGCCATAGTGCAGGATCACTAGCACCGCCTACATACCTCGCTCTGC SEQ ID NO: 32Reverse primer used in Example 43 to amplify 5 kb template from pCDNA3.1/CATAATAGCCGTTTTGTTTTTTGGATTACCAGTGTGCCATAGTGCAGGATCACCTATGTGGCGCGGTATTATCCCGTATTGAC SEQ ID NO: 33Reverse primer used in Example 43 to amplify 6.2 kb template from pCDNA3.1/CATAATAGCCGTTTTGTTTTTTGGATTACCAGTGTGCCATAGTGCAGGATCACATTTCGATAAGCCAGTAAGCAGTGGGTTCTCTAG SEQ ID NO: 34Forward qPCR primer used in Example 43 to detect linear transcript from pCDNA3.1/CAT ATTCTTGCCCGCCTGATGAA SEQ ID NO: 35Reverse qPCR primer used in Example 43 to detect linear transcript from pCDNA3.1/CAT TTGCTCATGGAAAACGGTGT SEQ ID NO: 36Forward qPCR primer used in Example 43 to detect circular transcript from pCDNA3.1/CAT TGATCCTGCACTATGGCACA SEQ ID NO: 37Reverse qPCR primer used in Example 43 to detect circular transcript from pCDNA3.1/CAT CTGGACTAGTGGATCCGAGC SEQ ID NO: 38Forward primer sequence used in Example 44 to detect ACTINGACGAGGCCCAGAGCAAGAGAGG SEQ ID NO: 39Reverse primer sequence used in Example 44 to detect ACTINGGTGTTGAAGGTCTCAAACATG SEQ ID NO: 40Forward primer sequence used in Example 44 to detect RIG-ITGTGGGCAATGTCATCAAAA SEQ ID NO: 41Reverse primer sequence used in Example 44 to detect RIG-IGAAGCACTTGCTACCTCTTGC SEQ ID NO: 42Forward primer sequence used in Example 44 to detect MDA5GGCACCATGGGAAGTGATT SEQ ID NO: 43Reverse primer sequence used in Example 44 to detect MDA5ATTTGGTAAGGCCTGAGCTG SEQ ID NO: 44Forward primer sequence used in Example 44 to detect PKRTCGCTGGTATCACTCGTCTG SEQ ID NO: 45Reverse primer sequence used in Example 44 to detect PKRGATTCTGAAGACCGCCAGAG SEQ ID NO: 46Forward primer sequence used in Example 44 to detect IFN-betaCTCTCCTGTTGTGCTTCTCC SEQ ID NO: 47Reverse primer sequence used in Example 44 to detect IFN-betaGTCAAAGTTCATCCTGTCCTTG. SEQ ID NO: 48EMCV T2A 3XFLAG-GFP F2A 3XFALG-Nluc P2A ISGGGAATAGCCGAAAAACAAAAAACAAAAAAAACAAAAAAAAAACCAAAAAAACAAAACACAACGTTACTGGCCGAAGCCGCTTGGAACAAGGCCGGTGTGCGTTTGTCTATATGTTATTTTCCACCATATTGCCGTCTTTTGGCAATGTGAGGGCCCGGAAACCTGGCCCTGTCTTCTTGACGAGCATTCCTAGGGGTCTTTCCCCTCTCGCCAAAGGAATGCAAGGTCTGTTGAATGTCGTGAAGGAAGCAGTTCCTCTGGAAGCTTCTTGTAGACAAACAACGTCTGTAGCGACCCTTTGCAGGCAGCGGAACCCCCCACCTGGCGACAGGTGCCTCTGCGGCCAAAAGCCACGTGTATACGATACACCTGCAAAGGCGGCACAACCCCAGTGCCACGTTGTGAGTTGGATAGTTGTGGAAAGAGTCAAATGGCTCTCCTCAAGCGTATTCAACAAGGGGCTGAAGGATGCCCAGAAGGTACCCCATTGTATGGGATCTGATCTGGGGCCTCGGTGCACATGCTTTACATGTGTTCAGTCGAGGTTAAAAAACGTCCAGGCCCCCCGAACCACGGGGACGTGGTTTTCCTTTGAAAAACACGATGATAATATGGCCACAACCATGGGCTCCGGCGAGGGCAGGGGAAGTCTTCTAACATGCGGGGACGTGGAGGAAAATCCCGGCCCAGACTACAAGGACGACGACGACAAGATCATCGACTATAAAGACGACGACGATAAAGGTGGCGACTATAAGGACGACGACGACAAAGCCATTGTGAGCAAGGGCGAGGAGCTGTTCACCGGGGTGGTGCCCATCCTGGTCGAGCTGGACGGCGACGTAAACGGCCACAAGTTCAGCGTGTCCGGCGAGGGCGAGGGCGATGCCACCTACGGCAAGCTGACCCTGAAGTTCATCTGCACCACCGGCAAGCTGCCCGTGCCCTGGCCCACCCTCGTGACCACCCTGACCTACGGCGTGCAGTGCTTCAGCCGCTACCCCGACCACATGAAGCAGCACGACTTCTTCAAGTCCGCCATGCCCGAAGGCTACGTCCAGGAGCGCACCATCTTCTTCAAGGACGACGGCAACTACAAGACCCGCGCCGAGGTGAAGTTCGAGGGCGACACCCTGGTGAACCGCATCGAGCTGAAGGGCATCGACTTCAAGGAGGACGGCAACATCCTGGGGCACAAGCTGGAGTACAACTACAACAGCCACAACGTCTATATCATGGCCGACAAGCAGAAGAACGGCATCAAGGTGAACTTCAAGATCCGCCACAACATCGAGGACGGCAGCGTGCAGCTCGCCGACCACTACCAGCAGAACACCCCCATCGGCGACGGCCCCGTGCTGCTGCCCGACAACCACTACCTGAGCACCCAGTCCGCCCTGAGCAAAGACCCCAACGAGAAGCGCGATCACATGGTCCTGCTGGAGTTCGTGACCGCCGCCGGGATCACTCTCGGCATGGACGAGCTGTACAAGGGAAGCGGAGTGAAACAGACTTTGAATTTTGACCTTCTCAAGTTGGCGGGAGACGTGGAGTCCAACCCTGGACCTGACTACAAGGACGACGACGACAAGATCATCGACTATAAAGACGACGACGATAAAGGTGGCGACTATAAGGACGACGACGACAAAGCCATTATCATCGTCTTCACACTCGAAGATTTCGTTGGGGACTGGCGACAGACAGCCGGCTACAACCTGGACCAAGTCCTTGAACAGGGAGGTGTGTCCAGTTTGTTTCAGAATCTCGGGGTGTCCGTAACTCCGATCCAAAGGATTGTCCTGAGCGGTGAAAATGGGCTGAAGATCGACATCCATGTCATCATCCCGTATGAAGGTCTGAGCGGCGACCAAATGGGCCAGATCGAAAAAATTTTTAAGGTGGTGTACCCTGTGGATGATCATCACTTTAAGGTGATCCTGCACTATGGCACACTGGTAATCGACGGGGTTACGCCGAACATGATCGACTATTTCGGACGGCCGTATGAAGGCATCGCCGTGTTCGACGGCAAAAAGATCACTGTAACAGGGACCCTGTGGAACGGCAACAAAATTATCGACGAGCGCCTGATCAACCCCGACGGCTCCCTGCTGTTCCGAGTAACCATCAACGGAGTGACCGGCTGGCGGCTGTGCGAACGCATTCTGGCGGGAAGCGGAGCTACTAACTTCAGCCTGCTGAAGCAGGCTGGAGACGTGGAGGAGAACCCTGGACCTTAAAAAAAACAAAAAACAAAACGGCTATT SEQ ID NO: 49EMCV T2A 3XFLAG-GFP F2A 3XFALG-Nluc P2A ISGGGAATAGCCGAAAAACAAAAAACAAAAAAAACAAAAAAAAAACCAAAAAAACAAAACACAACGTTACTGGCCGAAGCCGCTTGGAACAAGGCCGGTGTGCGTTTGTCTATATGTTATTTTCCACCATATTGCCGTCTTTTGGCAATGTGAGGGCCCGGAAACCTGGCCCTGTCTTCTTGACGAGCATTCCTAGGGGTCTTTCCCCTCTCGCCAAAGGAATGCAAGGTCTGTTGAATGTCGTGAAGGAAGCAGTTCCTCTGGAAGCTTCTTGTAGACAAACAACGTCTGTAGCGACCCTTTGCAGGCAGCGGAACCCCCCACCTGGCGACAGGTGCCTCTGCGGCCAAAAGCCACGTGTATACGATACACCTGCAAAGGCGGCACAACCCCAGTGCCACGTTGTGAGTTGGATAGTTGTGGAAAGAGTCAAATGGCTCTCCTCAAGCGTATTCAACAAGGGGCTGAAGGATGCCCAGAAGGTACCCCATTGTATGGGATCTGATCTGGGGCCTCGGTGCACATGCTTTACATGTGTTCAGTCGAGGTTAAAAAACGTCCAGGCCCCCCGAACCACGGGGACGTGGTTTTCCTTTGAAAAACACGATGATAATATGGCCACAACCATGGGCTCCGGCGAGGGCAGGGGAAGTCTTCTAACATGCGGGGACGTGGAGGAAAATCCCGGCCCAGACTACAAGGACGACGACGACAAGATCATCGACTATAAAGACGACGACGATAAAGGTGGCGACTATAAGGACGACGACGACAAAGCCATTGTGAGCAAGGGCGAGGAGCTGTTCACCGGGGTGGTGCCCATCCTGGTCGAGCTGGACGGCGACGTAAACGGCCACAAGTTCAGCGTGTCCGGCGAGGGCGAGGGCGATGCCACCTACGGCAAGCTGACCCTGAAGTTCATCTGCACCACCGGCAAGCTGCCCGTGCCCTGGCCCACCCTCGTGACCACCCTGACCTACGGCGTGCAGTGCTTCAGCCGCTACCCCGACCACATGAAGCAGCACGACTTCTTCAAGTCCGCCATGCCCGAAGGCTACGTCCAGGAGCGCACCATCTTCTTCAAGGACGACGGCAACTACAAGACCCGCGCCGAGGTGAAGTTCGAGGGCGACACCCTGGTGAACCGCATCGAGCTGAAGGGCATCGACTTCAAGGAGGACGGCAACATCCTGGGGCACAAGCTGGAGTACAACTACAACAGCCACAACGTCTATATCATGGCCGACAAGCAGAAGAACGGCATCAAGGTGAACTTCAAGATCCGCCACAACATCGAGGACGGCAGCGTGCAGCTCGCCGACCACTACCAGCAGAACACCCCCATCGGCGACGGCCCCGTGCTGCTGCCCGACAACCACTACCTGAGCACCCAGTCCGCCCTGAGCAAAGACCCCAACGAGAAGCGCGATCACATGGTCCTGCTGGAGTTCGTGACCGCCGCCGGGATCACTCTCGGCATGGACGAGCTGTACAAGGGAAGCGGAGTGAAACAGACTTTGAATTTTGACCTTCTCAAGTTGGCGGGAGACGTGGAGTCCAACCCTGGACCTTGATAGTAAGACTACAAGGACGACGACGACAAGATCATCGACTATAAAGACGACGACGATAAAGGTGGCGACTATAAGGACGACGACGACAAAGCCATTATCATCGTCTTCACACTCGAAGATTTCGTTGGGGACTGGCGACAGACAGCCGGCTACAACCTGGACCAAGTCCTTGAACAGGGAGGTGTGTCCAGTTTGTTTCAGAATCTCGGGGTGTCCGTAACTCCGATCCAAAGGATTGTCCTGAGCGGTGAAAATGGGCTGAAGATCGACATCCATGTCATCATCCCGTATGAAGGTCTGAGCGGCGACCAAATGGGCCAGATCGAAAAAATTTTTAAGGTGGTGTACCCTGTGGATGATCATCACTTTAAGGTGATCCTGCACTATGGCACACTGGTAATCGACGGGGTTACGCCGAACATGATCGACTATTTCGGACGGCCGTATGAAGGCATCGCCGTGTTCGACGGCAAAAAGATCACTGTAACAGGGACCCTGTGGAACGGCAACAAAATTATCGACGAGCGCCTGATCAACCCCGACGGCTCCCTGCTGTTCCGAGTAACCATCAACGGAGTGACCGGCTGGCGGCTGTGCGAACGCATTCTGGCGGGAAGCGGAGCTACTAACTTCAGCCTGCTGAAGCAGGCTGGAGACGTGGAGGAGAACCCTGGACCTTAAAAAAAACAAAAAACAAAACGGCTATT SEQ ID NO: 50 UREUCAUAAUCAAUUUAUUAUUUUCUUUUAUUUUAUUCACAUAAUUUUGUUUUU SEQ ID NO: 51 CSEAUUUUGUUUUUAACAUUUC SEQ ID NO: 52 URE/CSEUCAUAAUCAAUUUAUUAUUUUCUUUUAUUUUAUUCACAUAAUUUUGUUUUUAUUUUGUUUU UAACAUUUCSEQ ID NO: 53 CVB3-GLuc-STOP-UREAAAAUCCGUUGACCUUAAACGGUCGUGUGGGUUCAAGUCCCUCCACCCCCACGCCGGAAACGCAAUAGCCGAAAAACAAAAAACAAAAAAAACAAAAAAAAAACCAAAAAAACAAAACACAUUAAAACAGCCUGUGGGUUGAUCCCACCCACAGGCCCAUUGGGCGCUAGCACUCUGGUAUCACGGUACCUUUGUGCGCCUGUUUUAUACCCCCUCCCCCAACUGUAACUUAGAAGUAACACACACCGAUCAACAGUCAGCGUGGCACACCAGCCACGUUUUGAUCAAGCACUUCUGUUACCCCGGACUGAGUAUCAAUAGACUGCUCACGCGGUUGAAGGAGAAAGCGUUCGUUAUCCGGCCAACUACUUCGAAAAACCUAGUAACACCGUGGAAGUUGCAGAGUGUUUCGCUCAGCACUACCCCAGUGUAGAUCAGGUCGAUGAGUCACCGCAUUCCCCACGGGCGACCGUGGCGGUGGCUGCGUUGGCGGCCUGCCCAUGGGGAAACCCAUGGGACGCUCUAAUACAGACAUGGUGCGAAGAGUCUAUUGAGCUAGUUGGUAGUCCUCCGGCCCCUGAAUGCGGCUAAUCCUAACUGCGGAGCACACACCCUCAAGCCAGAGGGCAGUGUGUCGUAACGGGCAACUCUGCAGCGGAACCGACUACUUUGGGUGUCCGUGUUUCAUUUUAUUCCUAUACUGGCUGCUUAUGGUGACAAUUGAGAGAUCGUUACCAUAUAGCUAUUGGAUUGGCCAUCCGGUGACUAAUAGAGCUAUUAUAUAUCCCUUUGUUGGGUUUAUACCACUUAGCUUGAAAGAGGUUAAAACAUUACAAUUCAUUGUUAAGUUGAAUACAGCAAAAUGGGAGUCAAAGUUCUGUUUGCCCUGAUCUGCAUCGCUGUGGCCGAGGCCAAGCCCACCGAGAACAACGAAGACUUCAACAUCGUGGCCGUGGCCAGCAACUUCGCGACCACGGAUCUCGAUGCUGACCGCGGGAAGUUGCCCGGCAAGAAGCUGCCGCUGGAGGUGCUCAAAGAGAUGGAAGCCAAUGCCCGGAAAGCUGGCUGCACCAGGGGCUGUCUGAUCUGCCUGUCCCACAUCAAGUGCACGCCCAAGAUGAAGAAGUUCAUCCCAGGACGCUGCCACACCUACGAAGGCGACAAAGAGUCCGCACAGGGCGGCAUAGGCGAGGCGAUCGUCGACAUUCCUGAGAUUCCUGGGUUCAAGGACUUGGAGCCCAUGGAGCAGUUCAUCGCACAGGUCGAUCUGUGUGUGGACUGCACAACUGGCUGCCUCAAAGGGCUUGCCAACGUGCAGUGUUCUGACCUGCUCAAGAAGUGGCUGCCGCAACGCUGUGCGACCUUUGCCAGCAAGAUCCAGGGCCAGGUGGACAAGAUCAAGGGGGCCGGUGGUGACUAAUCAUAAUCAAUUUAUUAUUUUCUUUUAUUUUAUUCACAUAAUUUUGUUUUUAUUUUGUUUUUAACAUUUCAAAAAACAAAAAACAAAACGGCUAUUAUGCGUUACCGGCGAGACGCUACGGACUUSEQ ID NO: 54 CVB3-GLuc-STOP-URE/CSEAAAAUCCGUUGACCUUAAACGGUCGUGUGGGUUCAAGUCCCUCCACCCCCACGCCGGAAACGCAAUAGCCGAAAAACAAAAAACAAAAAAAACAAAAAAAAAACCAAAAAAACAAAACACAUUAAAACAGCCUGUGGGUUGAUCCCACCCACAGGCCCAUUGGGCGCUAGCACUCUGGUAUCACGGUACCUUUGUGCGCCUGUUUUAUACCCCCUCCCCCAACUGUAACUUAGAAGUAACACACACCGAUCAACAGUCAGCGUGGCACACCAGCCACGUUUUGAUCAAGCACUUCUGUUACCCCGGACUGAGUAUCAAUAGACUGCUCACGCGGUUGAAGGAGAAAGCGUUCGUUAUCCGGCCAACUACUUCGAAAAACCUAGUAACACCGUGGAAGUUGCAGAGUGUUUCGCUCAGCACUACCCCAGUGUAGAUCAGGUCGAUGAGUCACCGCAUUCCCCACGGGCGACCGUGGCGGUGGCUGCGUUGGCGGCCUGCCCAUGGGGAAACCCAUGGGACGCUCUAAUACAGACAUGGUGCGAAGAGUCUAUUGAGCUAGUUGGUAGUCCUCCGGCCCCUGAAUGCGGCUAAUCCUAACUGCGGAGCACACACCCUCAAGCCAGAGGGCAGUGUGUCGUAACGGGCAACUCUGCAGCGGAACCGACUACUUUGGGUGUCCGUGUUUCAUUUUAUUCCUAUACUGGCUGCUUAUGGUGACAAUUGAGAGAUCGUUACCAUAUAGCUAUUGGAUUGGCCAUCCGGUGACUAAUAGAGCUAUUAUAUAUCCCUUUGUUGGGUUUAUACCACUUAGCUUGAAAGAGGUUAAAACAUUACAAUUCAUUGUUAAGUUGAAUACAGCAAAAUGGGAGUCAAAGUUCUGUUUGCCCUGAUCUGCAUCGCUGUGGCCGAGGCCAAGCCCACCGAGAACAACGAAGACUUCAACAUCGUGGCCGUGGCCAGCAACUUCGCGACCACGGAUCUCGAUGCUGACCGCGGGAAGUUGCCCGGCAAGAAGCUGCCGCUGGAGGUGCUCAAAGAGAUGGAAGCCAAUGCCCGGAAAGCUGGCUGCACCAGGGGCUGUCUGAUCUGCCUGUCCCACAUCAAGUGCACGCCCAAGAUGAAGAAGUUCAUCCCAGGACGCUGCCACACCUACGAAGGCGACAAAGAGUCCGCACAGGGCGGCAUAGGCGAGGCGAUCGUCGACAUUCCUGAGAUUCCUGGGUUCAAGGACUUGGAGCCCAUGGAGCAGUUCAUCGCACAGGUCGAUCUGUGUGUGGACUGCACAACUGGCUGCCUCAAAGGGCUUGCCAACGUGCAGUGUUCUGACCUGCUCAAGAAGUGGCUGCCGCAACGCUGUGCGACCUUUGCCAGCAAGAUCCAGGGCCAGGUGGACAAGAUCAAGGGGGCCGGUGGUGACUAAUCAUAAUCAAUUUAUUAUUUUCUUUUAUUUUAUUCACAUAAUUUUGUUUUUAUUUUGUUUUUAACAUUUCAAAAAACAAAAAACAAAACGGCUAUUAUGCGUUACCGGCGAGACGCUACGGACUUSEQ ID NO: 55 Complementary primer used for example 42CACCGCTCAGGACAATCCTT SEQ ID NO: 56 CVB3 IRESTTAAAACAGCCTGTGGGTTGATCCCACCCACAGGCCCATTGGGCGCTAGCACTCTGGTATCACGGTACCTTTGTGCGCCTGTTTTATACCCCCTCCCCCAACTGTAACTTAGAAGTAACACACACCGATCAACAGTCAGCGTGGCACACCAGCCACGTTTTGATCAAGCACTTCTGTTACCCCGGACTGAGTATCAATAGACTGCTCACGCGGTTGAAGGAGAAAGCGTTCGTTATCCGGCCAACTACTTCGAAAAACCTAGTAACACCGTGGAAGTTGCAGAGTGTTTCGCTCAGCACTACCCCAGTGTAGATCAGGTCGATGAGTCACCGCATTCCCCACGGGCGACCGTGGCGGTGGCTGCGTTGGCGGCCTGCCCATGGGGAAACCCATGGGACGCTCTAATACAGACATGGTGCGAAGAGTCTATTGAGCTAGTTGGTAGTCCTCCGGCCCCTGAATGCGGCTAATCCTAACTGCGGAGCACACACCCTCAAGCCAGAGGGCAGTGTGTCGTAACGGGCAACTCTGCAGCGGAACCGACTACTTTGGGTGTCCGTGTTTCATTTTATTCCTATACTGGCTGCTTATGGTGACAATTGAGAGATCGTTACCATATAGCTATTGGATTGGCCATCCGGTGACTAATAGAGCTATTATATATCCCTTTGTTGGGTTTATACCACTTAGCTTGAAAGAGGTTAAAACATTACAATTCATTGTTAAGTTGAATACAGCAAA SEQ ID NO: 57 GlucATGGGAGTCAAAGTTCTGTTTGCCCTGATCTGCATCGCTGTGGCCGAGGCCAAGCCCACCGAGAACAACGAAGACTTCAACATCGTGGCCGTGGCCAGCAACTTCGCGACCACGGATCTCGATGCTGACCGCGGGAAGTTGCCCGGCAAGAAGCTGCCGCTGGAGGTGCTCAAAGAGATGGAAGCCAATGCCCGGAAAGCTGGCTGCACCAGGGGCTGTCTGATCTGCCTGTCCCACATCAAGTGCACGCCCAAGATGAAGAAGTTCATCCCAGGACGCTGCCACACCTACGAAGGCGACAAAGAGTCCGCACAGGGCGGCATAGGCGAGGCGATCGTCGACATTCCTGAGATTCCTGGGTTCAAGGACTTGGAGCCCATGGAGCAGTTCATCGCACAGGTCGATCTGTGTGTGGACTGCACAACTGGCTGCCTCAAAGGGCTTGCCAACGTGCAGTGTTCTGACCTGCTCAAGAAGTGGCTGCCGCAACGCTGTGCGACCTTTGCCAGCAAGATCCAGGGCCAGGTGGACAAGATCAAGGGGGCCGGTGGTGACTAA SEQ ID NO: 58EMCV IRES with stop mutationsACGTTACTGGCCGAAGCCGCTTGGAACAAGGCCGGTGTGCGTTTGTCTATATGTTATTTTCCACCATATTGCCGTCTTTTGGCAATGTGAGGGCCCGGAAACCTGGCCCTGTCTTCTTGACGAGCATTCCTAGGGGTCTTTCCCCTCTCGCCAAAGGAATGCAAGGTCTGTTGAATGTCGTGAAGGAAGCAGTTCCTCTGGAAGCTTCTTCAAGACAAACAACGTCTGTAGCGACCCTTTGCAGGCAGCGGAACCCCCCACCTGGCGACAGGTGCCTCTGCGGCCAAAAGCCACGTGTATACGATACACCTGCAAAGGCGGCACAACCCCAGTGCCACGTTGTGAGTTGGATAGTTGTGGAAAGAGTCAAATGGCTCTCCTCAAGCGTATTCAACAAGGGGCTGAAGGATGCCCAGAAGGTACCCCATTGTATGGGATCTGATCTGGGGCCTCGGTGCACATGCTTTACATGTGTTCAGTCGAGGTTAAAAAACGTCCAGGCCCCCCGAACCACGGGGACGTGGTTTTCCTTTGAAAAACACGATGATAATATGGCCACAACCATG SEQ ID NO: 59(SEQ ID NO: 59) SPACER 1AAAAUCCGUUGACCUUAAACGGUCGUGUGGGUUCAAGUCCCUCCACCCCCACGCCGGAAACGCAAUAGCCGAAAAACAAAAAACAAAAAAAACAAAAAAAAAACCAAAAAAACAAAACACA(SEQ ID NO: 135) SPACER 2AAAAAACAAAAAACAAAACGGCUAUUAUGCGUUACCGGCGAGACGCUACGGACUU SEQ ID: 60ATACCAGCCGAAAGGCCCTTGGCAGAGAGGTCTGAAAAGACCTCTGCTGACTATGTGATCTTATTAAAATTAGGTTAAATTTCGAGGTTAAAAATAGTTTTAATATTGCTATAGTCTTAGAGGTCTTGTATATTTATACTTACCACACAAGATGGACCGGAGCAGCCCTCCAATATCTAGTGTACCCTCGTGCTCGCTCAAACATTAAGTGGTGTTGTGCGAAAAGAATCTCACTTCAAGAAAAAGAAACTAGT

EMBODIMENT PARAGRAPHS

-   [1] A method of in vivo expression of one or more expression    sequences in a subject, comprising:    -   administering a circular polyribonucleotide to a cell of the        subject wherein the circular polyribonucleotide comprises the        one or more expression sequences; and    -   expressing the one or more expression sequences from the        circular polyribonucleotide in the cell, wherein the circular        polyribonucleotide is configured such that expression of the one        or more expression sequences in the cell at a later time point        is equal to or higher than an earlier time point.-   [2] A method of in vivo expression of one or more expression    sequences in a subject, comprising:    -   administering a circular polyribonucleotide to a cell of the        subject wherein the circular polyribonucleotide comprises the        one or more expression sequences; and    -   expressing the one or more expression sequences from the        circular polyribonucleotide in the cell, wherein the circular        polyribonucleotide is configured such that expression of the one        or more expression sequences in the cell over a time period of        at least 7, 8, 9, 10, 12, 14, or 16 days does not decrease by        greater than about 40%.-   [3] A method of in vivo expression of one or more expression    sequences in a subject, comprising:    -   administering a circular polyribonucleotide to a cell of the        subject wherein the circular polyribonucleotide comprises the        one or more expression sequences; and    -   expressing the one or more expression sequences from the        circular polyribonucleotide in the cell, wherein the circular        polyribonucleotide is configured such that expression of the one        or more expression sequences in the cell is maintained at a        level that does not vary by more than about 40% for at least 7,        8, 9, 10, 12, 14, or 16 days.-   [4] The method of any one of paragraphs [1] to [3], wherein    expression product of the one or more expression sequences comprises    a therapeutic protein.-   [5] The method of paragraph [4], wherein the therapeutic protein has    antioxidant activity, binding, cargo receptor activity, catalytic    activity, molecular carrier activity, molecular function regulator,    molecular transducer activity, nutrient reservoir activity, protein    tag, structural molecule activity, toxin activity, transcription    regulator activity, translation regulator activity, or transporter    activity.-   [6] The method of any one of paragraphs [1] to [5], Expression    product of the one or more expression sequences comprises a    secretary protein.-   [7] The method of paragraph [6], wherein the secretary protein    comprises a secretary enzyme.-   [8] The method of paragraph [6], wherein the secretary protein    comprises a secretary antibody.-   [9] The method of any one of paragraphs [1] to [8], wherein the    circular polyribonucleotide is at least about 20 nucleotides, at    least about 30 nucleotides, at least about 40 nucleotides, at least    about 50 nucleotides, at least about 75 nucleotides, at least about    100 nucleotides, at least about 200 nucleotides, at least about 300    nucleotides, at least about 400 nucleotides, at least about 500    nucleotides, at least about 1,000 nucleotides, at least about 2,000    nucleotides, at least about 5,000 nucleotides, at least about 6,000    nucleotides, at least about 7,000 nucleotides, at least about 8,000    nucleotides, at least about 9,000 nucleotides, at least about 10,000    nucleotides, at least about 12,000 nucleotides, at least about    14,000 nucleotides, at least about 15,000 nucleotides, at least    about 16,000 nucleotides, at least about 17,000 nucleotides, at    least about 18,000 nucleotides, at least about 19,000 nucleotides,    or at least about 20,000 nucleotides.-   [10] The method of any one of paragraphs [1] to [9], wherein the    circular polyribonucleotide has a persistence in the cell for at    least about 1 hr, 2 hrs, 6 hrs, 12 hrs, 18 hrs, 24 hrs, 2 days, 3,    days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11    days, 12 days, 13 days, 14 days, 15 days, 16 days, 17 days, 18 days,    19 days, 20 days, 21 days, 22 days, 23 days, 24 days, 25 days, 26    days, 27 days, 28 days, 29 days, 30 days, 60 days, or longer or any    time therebetween.-   [11] The method of any one of paragraphs [1] to [10], wherein the    circular polyribonucleotide has a persistence in the cell when the    cell is dividing for at least about 1 hr, 2 hrs, 6 hrs, 12 hrs, 18    hrs, 24 hrs, 2 days, 3, days, 4 days, 5 days, 6 days, 7 days, 8    days, 9 days, 10 days, 11 days, 12 days, 13 days, 14 days, 15 days,    16 days, 17 days, 18 days, 19 days, 20 days, 21 days, 22 days, 23    days, 24 days, 25 days, 26 days, 27 days, 28 days, 29 days, 30 days,    60 days, or longer or any time therebetween.-   [12] The method of any one of paragraphs [1] to [11], wherein the    circular polyribonucleotide has a persistence in the cell post    division for at least about 1 hr, 2 hrs, 6 hrs, 12 hrs, 18 hrs, 24    hrs, 2 days, 3, days, 4 days, 5 days, 6 days, 7 days, 8 days, 9    days, 10 days, 11 days, 12 days, 13 days, 14 days, 15 days, 16 days,    17 days, 18 days, 19 days, 20 days, 21 days, 22 days, 23 days, 24    days, 25 days, 26 days, 27 days, 28 days, 29 days, 30 days, 60 days,    or longer or any time therebetween.-   [13] The method of any one of paragraphs [1] to [12], wherein the    circular polyribonucleotide comprises at least one spacer sequence.-   [14] The method of paragraph [13], wherein the spacer sequence is    configured to provide conformational flexibility between elements of    the circular polyribonucleotide on both sides of the spacer    sequence.-   [15] The method of paragraph [13] or [14], wherein a ratio of the    spacer sequence to a non-spacer sequence of the circular    polyribonucleotide, e.g., expression sequences, of about 0.05:1,    about 0.06:1, about 0.07:1,about 0.08:1, about 0.09:1, about 0.1:1,    about 0.12:1, about 0.125:1, about 0.15:1, about 0.175:1, about    0.2:1, about 0.225:1, about 0.25:1, about 0.3:1, about 0.35:1, about    0.4:1, about 0.45:1, about 0.5:1, about 0.55:1, about 0.6:1, about    0.65:1, about 0.7:1, about 0.75:1, about 0.8:1, about 0.85:1, about    0.9:1, about 0.95:1, about 0.98:1, about 1:1, about 1.02:1, about    1.05:1, about 1.1:1, about 1.15:1, about 1.2:1, about 1.25:1, about    1.3:1, about 1.35:1, about 1.4:1, about 1.45:1, about 1.5:1, about    1.55:1, about 1.6:1, about 1.65:1, about 1.7:1, about 1.75:1, about    1.8:1, about 1.85:1, about 1.9:1, about 1.95:1, about 1.975:1, about    1.98:1, or about 2:1.-   [16] The method of any one of paragraphs [13] to [15], wherein the    spacer sequence comprises at least 3 ribonucleotides, at least 4    ribonucleotides, at least 5 ribonucleotides, at least about 8    ribonucleotides, at least about 10 ribonucleotides, at least about    12 ribonucleotides, at least about 15 ribonucleotides, at least    about 20 ribonucleotides, at least about 25 ribonucleotides, at    least about 30 ribonucleotides, at least about 40 ribonucleotides,    at least about 50 ribonucleotides, at least about 60    ribonucleotides, at least about 70 ribonucleotides, at least about    80 ribonucleotides, at least about 90 ribonucleotides, at least    about 100 ribonucleotides, at least about 120 ribonucleotides, at    least about 150 ribonucleotides, at least about 200 ribonucleotides,    at least about 250 ribonucleotides, at least about 300    ribonucleotides, at least about 400 ribonucleotides, at least about    500 ribonucleotides, at least about 600 ribonucleotides, at least    about 700 ribonucleotides, at least about 800 ribonucleotides, at    least about 900 ribonucleotides, or at least about 100    ribonucleotides.-   [17] The method of any one of paragraphs [13] to [16], wherein the    spacer sequence comprises at least 95%, 90%, 85%, 80%, 75%, 70%,    65%, 60%, 55%, 50%, 55%, 50%, 45%, 40%, 35%, 30%, 20% or any    percentage therebetween of adenine ribonucleotides.-   [18] The method of any one of paragraphs [1] to [17], wherein the    circular polyribonucleotide is competent for rolling translation.-   [19] The method of paragraph [18], wherein each of the one or more    expression sequences is separated from a succeeding expression    sequence by a stagger element on the circular polyribonucleotide,    wherein the rolling circle translation of the one or more expression    sequences generates at least two polypeptide molecules.-   [20] The method of paragraph [19], wherein the stagger element    prevents generation of a single polypeptide (a) from two rounds of    translation of a single expression sequence or (b) from one or more    rounds of translation of two or more expression sequences.-   [21] The method of paragraph [19] or [20], wherein the stagger    element is a sequence separate from the one or more expression    sequences.-   [22] The method of paragraph [19] or [20], wherein the stagger    element comprises a portion of an expression sequence of the one or    more expression sequences.-   [23] The method of any one of paragraphs [18] to [22], wherein the    circular polyribonucleotide is configured such that at least 50%, at    least 60%, at least 70%, at least 80%, at least 90%, at least 95%,    at least 96%, at least 97%, at least 98%, at least 99%, or 100% of    total polypeptides (molar/molar) generated during the rolling circle    translation of the circular polyribonucleotide are discrete    polypeptides, and wherein each of the discrete polypeptides is    generated from a single round of translation or less than a single    round of translation of the one or more expression sequences.-   [24] The method of paragraph [23], wherein the circular    polyribonucleotide is configured such that at least 50%, at least    60%, at least 70%, at least 80%, at least 90%, at least 95%, at    least 96%, at least 97%, at least 98%, at least 99%, or 100% of    total polypeptides (molar/molar) generated during the rolling circle    translation of the circular polyribonucleotide are the discrete    polypeptides, and wherein amount ratio of the discrete products over    the total polypeptides is tested in an in vitro translation system.-   [25] The method of paragraph [24], wherein the in vitro translation    system comprises rabbit reticulocyte lysate.-   [26] The method of any one of paragraphs [19] to [25], wherein the    stagger element is at a 3′ end of at least one of the one or more    expression sequences, and wherein the stagger element is configured    to stall a ribosome during rolling circle translation of the    circular polyribonucleotide.-   [27] The method of any one of paragraphs [19] to [26], wherein the    stagger element encodes a peptide sequence selected from the group    consisting of a 2A sequence and a 2A-like sequence.-   [28] The method of any one of paragraphs [19] to [27], wherein the    stagger element encodes a sequence with a C-terminal sequence that    is GP.-   [29] The method of any one of paragraphs [19] to [28], wherein the    stagger element encodes a sequence with a C-terminal consensus    sequence that is D(V/I)ExNPGP (SEQ ID NO: 61), where x=any amino    acid.-   [30] The method of any one of paragraphs [19] to [29], wherein the    stagger element encodes a sequence selected from the group    consisting of GDVESNPGP (SEQ ID NO: 62), GDIEENPGP (SEQ ID NO: 63),    VEPNPGP (SEQ ID NO: 64), IETNPGP (SEQ ID NO: 65), GDIESNPGP (SEQ ID    NO: 66), GDVELNPGP (SEQ ID NO: 67), GDIETNPGP (SEQ ID NO: 68),    GDVENPGP (SEQ ID NO: 69), GDVEENPGP (SEQ ID NO: 70), GDVEQNPGP (SEQ    ID NO: 71), IESNPGP (SEQ ID NO: 72), GDIELNPGP (SEQ ID NO: 73),    HDIETNPGP (SEQ ID NO: 74), HDVETNPGP (SEQ ID NO: 75), HDVEMNPGP (SEQ    ID NO: 76), GDMESNPGP (SEQ ID NO: 77), GDVETNPGP (SEQ ID NO: 78),    GDIEQNPGP (SEQ ID NO: 79), and DSEFNPGP (SEQ ID NO: 80).-   [31] The method of any one of paragraphs [19] to [30], wherein the    stagger element is at 3′ end of each of the one or more expression    sequences.-   [32] The method of any one of paragraphs [19] to [31], wherein the    stagger element of a first expression sequence in the circular    polyribonucleotide is upstream of (5′ to) a first translation    initiation sequence of an expression sequence succeeding the first    expression sequence in the circular polyribonucleotide, and wherein    a distance between the stagger element and the first translation    initiation sequence enables continuous translation of the first    expression sequence and the succeeding expression sequence.-   [33] The method of any one of paragraphs [19] to [32], wherein the    stagger element of a first expression sequence in the circular    polyribonucleotide is upstream of (5′ to) a first translation    initiation sequence of an expression sequence succeeding the first    expression in the circular polyribonucleotide, wherein the circular    polyribonucleotide is continuously translated, wherein a    corresponding circular polyribonucleotide comprising a second    stagger element upstream of a second translation initiation sequence    of a second expression sequence in the corresponding circular    polyribonucleotide is not continuously translated, and wherein the    second stagger element in the corresponding circular    polyribonucleotide is at a greater distance from the second    translation initiation sequence, e.g., at least 2×, 3×, 4×, 5×, 6×,    7×, 8×, 9×, 10×, than a distance between the stagger element and the    first translation initiation in the circular polyribonucleotide.-   [34] The method of any one of paragraphs [19] to [33], wherein the    distance between the stagger element and the first translation    initiation is at least 2 nt, 3 nt, 4 nt, 5 nt, 6 nt, 7 nt, 8 nt, 9    nt, 10 nt, 11 nt, 12 nt, 13 nt, 14 nt, 15 nt, 16 nt, 17 nt, 18 nt,    19 nt, 20 nt, 25 nt, 30 nt, 35 nt, 40 nt, 45 nt, 50 nt, 55 nt, 60    nt, 65 nt, 70 nt, 75 nt, or greater.-   [35] The method of any one of paragraphs [19] to [34], wherein the    distance between the second stagger element and the second    translation initiation is at least 2 nt, 3 nt, 4 nt, 5 nt, 6 nt, 7    nt, 8 nt, 9 nt, 10 nt, 11 nt, 12 nt, 13 nt, 14 nt, 15 nt, 16 nt, 17    nt, 18 nt, 19 nt, 20 nt, 25 nt, 30 nt, 35 nt, 40 nt, 45 nt, 50 nt,    55 nt, 60 nt, 65 nt, 70 nt, 75 nt, or greater than the distance    between the tagger element and the first translation initiation.-   [36] The method of any one of paragraphs [19] to [35], wherein the    expression sequence succeeding the first expression sequence on the    circular polyribonucleotide is an expression sequence other than the    first expression sequence.-   [37] The method of any one of paragraphs [19] to [35], wherein the    succeeding expression sequence of the first expression sequence on    the circular polyribonucleotide is the first expression sequence.-   [38] The method of any one of paragraphs [1] to [37], wherein the    circular polyribonucleotide comprises at least one structural    element selected from:    -   a) an encryptogen;    -   b) a stagger element;    -   c) a regulatory element;    -   d) a replication element; and    -   f) quasi-double-stranded secondary structure.-   [39] The method of any one of paragraphs [1] to [38], wherein the    circular polyribonucleotide comprises at least one functional    characteristic selected from:    -   a) greater translation efficiency than a linear counterpart;    -   b) a stoichiometric translation efficiency of multiple        translation products;    -   c) less immunogenicity than a counterpart lacking an        encryptogen;    -   d) increased half-life over a linear counterpart; and    -   e) persistence during cell division.-   [40] The method of any one of paragraphs [1] to [39], wherein the    circular polyribonucleotide has a translation efficiency at least    5%, at least 10%, at least 15%, at least 20%, at least 30%, at least    40%, at least 50%, at least 60%, at least 70%, at least 80%, at    least 90%, at least 100%, at least 150%, at least 2 fold, at least 3    fold, at least 4 fold, at least 5 fold, at least 6 fold, at least 7    fold, at least 8 fold, at least 9 fold, at least 10 fold, at least    20 fold, at least 50 fold, or at least 100 fold greater than a    linear counterpart.-   [41] The method of any one of paragraphs [1] to [40], wherein the    circular polyribonucleotide has a translation efficiency at least 5    fold greater than a linear counterpart.-   [42] The method of any one of paragraphs [1] to [41], wherein the    circular polyribonucleotide lacks at least one of:    -   a) a 5′-UTR;    -   b) a 3′-UTR;    -   c) a poly-A sequence;    -   d) a 5′-cap;    -   e) a termination element;    -   f) an internal ribosomal entry site;    -   g) degradation susceptibility by exonucleases; and    -   h) binding to a cap-binding protein.-   [43] The method of any one of [1] to [42], wherein the circular    polyribonucleotide comprises an internal ribosomal entry site.-   [44] The method of any one of [1] to [42], wherein the circular    polyribonucleotide lacks an internal ribosomal entry site.-   [45] The method of any one of [1] to [43], wherein the one or more    expression sequences comprise a Kozak initiation sequence.-   [46] The method of any one of [38] to [45], wherein the    quasi-helical structure comprises at least one double-stranded RNA    segment with at least one non-double-stranded segment.-   [47] The method of paragraph [38] or [46], wherein the quasi-helical    structure comprises a first sequence and a second sequence linked    with a repetitive sequence, e.g., an A-rich sequence.-   [48] The method of any one of paragraphs [38] to [47], wherein the    encryptogen comprises a splicing element.-   [49] The method of any one of paragraphs [38] to [48], wherein the    circular polyribonucleotide comprises at least one modified    ribonucleotide.-   [50] The method of any one of paragraphs [38] to [49], wherein the    circular polyribonucleotide comprises modified ribonucleotides in a    portion of its entire length.-   [51] The method of any one of paragraphs [38] to [50], wherein the    encryptogen comprises at least one modified ribonucleotide, e.g.,    pseudo-uridine, N(6)methyladenosine (m6A).-   [52] The method of any one of paragraphs [38] to [51], wherein the    encryptogen comprises a protein binding site, e.g., ribonucleotide    binding protein.-   [53] The method of any one of paragraphs [38] to [52], wherein the    encryptogen comprises an immunoprotein binding site, e.g., to evade    Immune reponses, e.g., CTL responses.-   [54] The method of any one of paragraphs [38] to [53], wherein the    circular polyribonucleotide has at least 2× less immunogenicity than    a counterpart lacking the encryptogen, e.g., as assessed by    expression or signaling or activation of at least one of RIG-I,    TLR-3, TLR-7, TLR-8, MDA-5, LGP-2, OAS, OASL, PKR, IFN-beta.-   [55] The method of any one of paragraphs [38] to [54], wherein the    circular polyribonucleotide further comprises a riboswitch.-   [56] The method of any one of paragraphs [38] to [55], wherein the    circular polyribonucleotide further comprises an aptazyme.-   [57] The method of any one of paragraphs [38] to [56], wherein the    circular polyribonucleotide comprises a translation initiation    sequence, e.g., GUG, CUG start codon, e.g., expression under stress    conditions.-   [58] The method of any one of paragraphs [38] to [57], wherein the    circular polyribonucleotide comprises a stagger element, e.g., 2A.-   [59] The method of any one of paragraphs [38] to [58], wherein the    circular polyribonucleotide comprises a regulatory nucleic acid,    e.g., a non-coding RNA.-   [60] The method of any one of paragraphs [38] to [59], wherein the    circular polyribonucleotide has a size in the range of about 20    bases to about 20 kb.-   [61] The method of any one of paragraphs [38] to [60], wherein the    circular polyribonucleotide is synthesized through circularization    of a linear polynucleotide.-   [62] The method of any one of paragraphs [38] to [61], wherein the    circular polyribonucleotide comprises a plurality of expression    sequences, either the same or different.-   [63] The method of any one of paragraphs [38] to [62], wherein the    circular polyribonucleotide is substantially resistant to    degradation, e.g., exonuclease.-   [64] The method of any one of paragraphs [38] to [63], wherein the    circular polyribonucleotide lacks at least one of:    -   a) a 5′-UTR;    -   b) a 3′-UTR;    -   c) a poly-A sequence;    -   d) a 5′-cap;    -   e) a termination element;    -   f) an internal ribosomal entry site;    -   g) degradation susceptibility by exonucleases; and    -   h) binding to a cap-binding protein.-   [65] A pharmaceutical composition comprising a circular    polyribonucleotide that comprises at least one structural element    selected from:    -   a) an encryptogen;    -   b) a stagger element;    -   c) a regulatory element;    -   d) a replication element;    -   f) quasi-double-stranded secondary structure; and    -   g) expression sequence;

and at least one functional characteristic selected from:

-   -   a) greater translation efficiency than a linear counterpart;    -   b) a stoichiometric translation efficiency of multiple        translation products;    -   c) less immunogenicity than a counterpart lacking an        encryptogen;    -   d) increased half-life over a linear counterpart; and    -   e) persistence during cell division.

-   [66] The composition of paragraph [65], wherein the circular    polyribonucleotide is translation competent.

-   [67] The composition of paragraph [65] or [66], wherein the    quasi-helical structure comprises at least one double-stranded RNA    segment with at least one non-double-stranded segment.

-   [68] The composition of any one of paragraphs [65] to [67], wherein    the quasi-helical structure comprises a first sequence and a second    sequence linked with a repetitive sequence, e.g., an A-rich    sequence.

-   [69] The composition of any one of paragraphs [65] to [68], wherein    the encryptogen comprises a splicing element.

-   [70] The composition of any one of paragraphs [65] to [69], wherein    the circular polyribonucleotide comprises at least one modified    ribonucleotide.

-   [71] The composition of any one of paragraphs [65] to [70], wherein    the encryptogen comprises at least one modified ribonucleotide,    e.g., pseudo-uridine, N(6)methyladenosine (m6A).

-   [72] The composition of any one of paragraphs [65] to [71], wherein    the encryptogen comprises a protein binding site, e.g.,    ribonucleotide binding protein.

-   [73] The composition of any one of paragraphs [65] to [72], wherein    the encryptogen comprises an immunoprotein binding site, e.g., to    evade Immune reponses, e.g., CTL responses.

-   [74] The composition of any one of paragraphs [65] to [73], wherein    the circular polyribonucleotide has at least 2× less immunogenicity    than a counterpart lacking the encryptogen, e.g., as assessed by    expression or signaling or activation of at least one of RIG-I,    TLR-3, TLR-7, TLR-8, MDA-5, LGP-2, OAS, OASL, PKR, IFN-beta.

-   [75] The composition of any one of paragraphs [65] to [74], wherein    the circular polyribonucleotide further comprises a riboswitch.

-   [76] The composition of any one of paragraphs [65] to [75], wherein    the circular polyribonucleotide further comprises an aptazyme.

-   [77] The composition of any one of paragraphs [65] to [76], wherein    the circular polyribonucleotide comprises a translation initiation    sequence, e.g., GUG, CUG start codon, e.g., expression under stress    conditions.

-   [78] The composition of any one of paragraphs [65] to [77], wherein    the circular polyribonucleotide comprises at least one expression    sequence, e.g., encoding a polypeptide.

-   [79] The composition of paragraph [78], wherein the expression    sequence encodes a peptide or polynucleotide.

-   [80] The composition of any one of paragraphs [65] to [79], wherein    the circular polyribonucleotide comprises a stagger element, e.g.,    2A.

-   [81] The composition of any one of paragraphs [65] to [80], wherein    the circular polyribonucleotide comprises a regulatory nucleic acid,    e.g., a non-coding RNA.

-   [82] The composition of any one of paragraphs [65] to [81], wherein    the circular polyribonucleotide has a size in the range of about 20    bases to about 20 kb.

-   [83] The composition of any one of paragraphs [65] to [82], wherein    the circular polyribonucleotide is synthesized through    circularization of a linear polynucleotide.

-   [84] The composition of any one of paragraphs [65] to [83], wherein    the circular polyribonucleotide comprises a plurality of expression    sequences, either the same or different.

-   [85] The composition of any one of paragraphs [65] to [84], wherein    the circular polyribonucleotide is substantially resistant to    degradation, e.g., exonuclease.

-   [86] The composition of any one of paragraphs [65] to [85], wherein    the circular polyribonucleotide lacks at least one of:    -   a) a 5′-UTR;    -   b) a 3′-UTR;    -   c) a poly-A sequence;    -   d) a 5′-cap;    -   e) a termination element;    -   f) an internal ribosomal entry site;    -   g) degradation susceptibility by exonucleases; and    -   h) binding to a cap-binding protein.

-   [87] A method of producing the composition of any one of paragraphs    [65] to [86] comprising combining the circular polyribonucleotide    with a pharmaceutically acceptable carrier or excipient.

-   [88] A method of treatment comprising administering the composition    of any one of paragraphs [65] to [86].

-   [89] A method for protein expression, comprising translating at    least a region of the circular polyribonucleotide of any one of    paragraphs [65] to [86].

-   [90] The method of paragraph [89], wherein the translation of the at    least a region of the circular polyribonucleotide takes place in    vitro.

-   [91] The method of paragraph [89], wherein the translation of the at    least a region of the circular polyribonucleotide takes place in    vivo.

-   [92] A polynucleotide encoding the circular polyribonucleotide of    any one of paragraphs [65] to [86].

-   [93] A pharmaceutical composition comprising a pharmaceutically    acceptable carrier or excipient and a circular polyribonucleotide    that comprises one or more expression sequences, wherein the    circular polyribonucleotide is competent for rolling circle    translation.

-   [94] The composition of paragraph [93], wherein each of the one or    more expression sequences is separated from a succeeding expression    sequence by a stagger element on the circular polyribonucleotide,    wherein the rolling circle translation of the one or more expression    sequences generates at least two polypeptide molecules.

-   [95] The composition of paragraph [94], wherein the stagger element    prevents generation of a single polypeptide (a) from two rounds of    translation of a single expression sequence or (b) from one or more    rounds of translation of two or more expression sequences.

-   [96] The composition of paragraph [94] or [95], wherein the stagger    element is a sequence separate from the one or more expression    sequences.

-   [97] The composition of paragraph [94] or [95], wherein the stagger    element comprises a portion of an expression sequence of the one or    more expression sequences.

-   [98] A pharmaceutical composition comprising a pharmaceutically    acceptable carrier or excipient and a circular polyribonucleotide    that comprises one or more expression sequences and is competent for    rolling circle translation, wherein the circular polyribonucleotide    is configured such that at least 50%, at least 60%, at least 70%, at    least 80%, at least 90%, at least 95%, at least 96%, at least 97%,    at least 98%, at least 99%, or 100% of total polypeptides    (molar/molar) generated during the rolling circle translation of the    circular polyribonucleotide are discrete polypeptides, and wherein    each of the discrete polypeptides is generated from a single round    of translation or less than a single round of translation of the one    or more expression sequences.

-   [99] The composition of paragraph [98], wherein the circular    polyribonucleotide is configured such that at least 50%, at least    60%, at least 70%, at least 80%, at least 90%, at least 95%, at    least 96%, at least 97%, at least 98%, at least 99%, or 100% of    total polypeptides (molar/molar) generated during the rolling circle    translation of the circular polyribonucleotide are the discrete    polypeptides, and wherein amount ratio of the discrete products over    the total polypeptides is tested in an in vitro translation system.

-   [100] The composition of paragraph [99], wherein the in vitro    translation system comprises rabbit reticulocyte lysate.

-   [101] The composition of any one of paragraphs [93] to [100],    wherein the stagger element is at a 3′ end of at least one of the    one or more expression sequences, and wherein the stagger element is    configured to stall a ribosome during rolling circle translation of    the circular polyribonucleotide.

-   [102] A pharmaceutical composition comprising a pharmaceutically    acceptable carrier or excipient and a circular polyribonucleotide    that comprises one or more expression sequences and a stagger    element at 3′ end of at least one of the one or more expression    sequences, wherein the stagger element is configured to stall a    ribosome during rolling circle translation of the circular    polyribonucleotide.

-   [103] The composition of paragraph [101] or [102], wherein the    stagger element encodes a peptide sequence selected from the group    consisting of a 2A sequence and a 2A-like sequence.

-   [104] The composition of any one of paragraph [101] to [103],    wherein the stagger element encodes a sequence with a C-terminal    sequence that is GP.

-   [105] The composition of any one of paragraph [101] to [104],    wherein the stagger element encodes a sequence with a C-terminal    consensus sequence that is D(V/I)ExNPGP (SEQ ID NO: 61), where x=any    amino acid.

-   [106] The composition of any one of paragraph [101] to [105],    wherein the stagger element encodes a sequence selected from the    group consisting of GDVESNPGP (SEQ ID NO: 62), GDIEENPGP (SEQ ID NO:    63), VEPNPGP (SEQ ID NO: 64), IETNPGP (SEQ ID NO: 65), GDIESNPGP    (SEQ ID NO: 66), GDVELNPGP (SEQ ID NO: 67), GDIETNPGP (SEQ ID NO:    68), GDVENPGP (SEQ ID NO: 69), GDVEENPGP (SEQ ID NO: 70), GDVEQNPGP    (SEQ ID NO: 71), IESNPGP (SEQ ID NO: 72), GDIELNPGP (SEQ ID NO: 73),    HDIETNPGP (SEQ ID NO: 74), HDVETNPGP (SEQ ID NO: 75), HDVEMNPGP (SEQ    ID NO: 76), GDMESNPGP (SEQ ID NO: 77), GDVETNPGP (SEQ ID NO: 78),    GDIEQNPGP (SEQ ID NO: 79), and DSEFNPGP (SEQ ID NO: 80).

-   [107] The composition of any one of paragraphs [101] to [106],    wherein the stagger element is at 3′ end of each of the one or more    expression sequences.

-   [108] The composition of any one of paragraphs [93] to [107],    wherein the stagger element of a first expression sequence in the    circular polyribonucleotide is upstream of (5′ to) a first    translation initiation sequence of an expression sequence succeeding    the first expression sequence in the circular polyribonucleotide,    and wherein a distance between the stagger element and the first    translation initiation sequence enables continuous translation of    the first expression sequence and the succeeding expression    sequence.

-   [109] The composition of any one of paragraphs [93] to [107],    wherein the stagger element of a first expression sequence in the    circular polyribonucleotide is upstream of (5′ to) a first    translation initiation sequence of an expression sequence succeeding    the first expression in the circular polyribonucleotide, wherein the    circular polyribonucleotide is continuously translated, wherein a    corresponding circular polyribonucleotide comprising a second    stagger element upstream of a second translation initiation sequence    of a second expression sequence in the corresponding circular    polyribonucleotide is not continuously translated, and wherein the    second stagger element in the corresponding circular    polyribonucleotide is at a greater distance from the second    translation initiation sequence, e.g., at least 2×, 3×, 4×, 5×, 6×,    7×, 8×, 9×, 10×, than a distance between the stagger element and the    first translation initiation in the circular polyribonucleotide.

-   [110] The composition of paragraph [108] or [109], wherein the    distance between the stagger element and the first translation    initiation is at least 2 nt, 3 nt, 4 nt, 5 nt, 6 nt, 7 nt, 8 nt, 9    nt, 10 nt, 11 nt, 12 nt, 13 nt, 14 nt, 15 nt, 16 nt, 17 nt, 18 nt,    19 nt, 20 nt, 25 nt, 30 nt, 35 nt, 40 nt, 45 nt, 50 nt, 55 nt, 60    nt, 65 nt, 70 nt, 75 nt, or greater.

-   [111] The composition of paragraph [108] or [109], wherein the    distance between the second stagger element and the second    translation initiation is at least 2 nt, 3 nt, 4 nt, 5 nt, 6 nt, 7    nt, 8 nt, 9 nt, 10 nt, 11 nt, 12 nt, 13 nt, 14 nt, 15 nt, 16 nt, 17    nt, 18 nt, 19 nt, 20 nt, 25 nt, 30 nt, 35 nt, 40 nt, 45 nt, 50 nt,    55 nt, 60 nt, 65 nt, 70 nt, 75 nt, or greater than the distance    between the tagger element and the first translation initiation.

-   [112] The composition of any one of paragraphs [108] to [110],    wherein the expression sequence succeeding the first expression    sequence on the circular polyribonucleotide is an expression    sequence other than the first expression sequence.

-   [113] The composition of any one of paragraphs [108] to [110],    wherein the succeeding expression sequence of the first expression    sequence on the circular polyribonucleotide is the first expression    sequence.

-   [114] The composition of any one of paragraphs [93] to [113],    wherein the circular polyribonucleotide comprises at least one    structural element selected from:    -   a) an encryptogen;    -   b) a stagger element;    -   c) a regulatory element;    -   d) a replication element; and    -   f) quasi-double-stranded secondary structure.

-   [115] The composition of any one of paragraphs [93] to [114],    wherein the circular polyribonucleotide comprises at least one    functional characteristic selected from:    -   a) greater translation efficiency than a linear counterpart;    -   b) a stoichiometric translation efficiency of multiple        translation products;    -   c) less immunogenicity than a counterpart lacking an        encryptogen;    -   d) increased half-life over a linear counterpart; and    -   e) persistence during cell division.

-   [116] The composition of any one of paragraphs [93] to [115],    wherein the circular polyribonucleotide has a translation efficiency    at least 5%, at least 10%, at least 15%, at least 20%, at least 30%,    at least 40%, at least 50%, at least 60%, at least 70%, at least    80%, at least 90%, at least 100%, at least 150%, at least 2 fold, at    least 3 fold, at least 4 fold, at least 5 fold, at least 6 fold, at    least 7 fold, at least 8 fold, at least 9 fold, at least 10 fold, at    least 20 fold, at least 50 fold, or at least 100 fold greater than a    linear counterpart.

-   [117] The composition of any one of paragraphs [93] to [115],    wherein the circular polyribonucleotide has a translation efficiency    at least 5 fold greater than a linear counterpart.

-   [118] The composition of any one of paragraphs [93] to [117],    wherein the circular polyribonucleotide lacks at least one of:    -   a) a 5′-UTR;    -   b) a 3′-UTR;    -   c) a poly-A sequence;    -   d) a 5′-cap;    -   e) a termination element;    -   f) an internal ribosomal entry site;    -   g) degradation susceptibility by exonucleases; and    -   h) binding to a cap-binding protein.

-   [119] The composition of any one of paragraphs[93] to [118], wherein    the circular polyribonucleotide lacks an internal ribosomal entry    site.

-   [120] The composition of any one of paragraphs [93] to [119],    wherein the one or more expression sequences comprise a Kozak    initiation sequence.

-   [121] The composition of any one of paragraphs [114] to [120],    wherein the quasi-helical structure comprises at least one    double-stranded RNA segment with at least one non-double-stranded    segment.

-   [122] The composition of paragraph [121], wherein the quasi-helical    structure comprises a first sequence and a second sequence linked    with a repetitive sequence, e.g., an A-rich sequence.

-   [123] The composition of any one of paragraphs [114] to [122],    wherein the encryptogen comprises a splicing element.

-   [124] The composition of any one of paragraphs [93] to [123],    wherein the circular polyribonucleotide comprises at least one    modified ribonucleotide.

-   [125] The composition of any one of paragraphs [93] to [124],    wherein the encryptogen comprises at least one modified    ribonucleotide, e.g., pseudo-uridine, N(6)methyladenosine (m6A).

-   [126] The composition of any one of paragraphs [93] to [125],    wherein the encryptogen comprises a protein binding site, e.g.,    ribonucleotide binding protein.

-   [127] The composition of any one of paragraphs [93] to [126],    wherein the encryptogen comprises an immunoprotein binding site,    e.g., to evade immune reponses, e.g., CTL responses.

-   [128] The composition of any one of paragraphs [93] to [127],    wherein the circular polyribonucleotide has at least 2× less    immunogenicity than a counterpart lacking the encryptogen, e.g., as    assessed by expression or signaling or activation of at least one of    RIG-I, TLR-3, TLR-7, TLR-8, MDA-5, LGP-2, OAS, OASL, PKR, IFN-beta.

-   [129] The composition of any one of paragraphs [93] to [128],    wherein the circular polyribonucleotide further comprises a    riboswitch.

-   [130] The composition of any one of paragraphs [93] to [129],    wherein the circular polyribonucleotide further comprises an    aptazyme.

-   [131] The composition of any one of paragraphs [93] to [130],    wherein the circular polyribonucleotide comprises a non-canonical    translation initiation sequence, e.g., GUG, CUG start codon, e.g., a    translation initiation sequence that initiates expression under    stress conditions.

-   [132] The composition of any one of paragraphs [93] to [131],    wherein the one or more expression sequences encodes a peptide.

-   [133] The composition of any one of paragraphs [93] to [132],    wherein the circular polyribonucleotide comprises a regulatory    nucleic acid, e.g., a non-coding RNA.

-   [134] The composition of any one of paragraphs [93] to [133],    wherein the circular polyribonucleotide has a size in the range of    about 20 bases to about 20 kb.

-   [135] The composition of any one of paragraphs [93] to [134],    wherein the circular polyribonucleotide is synthesized through    circularization of a linear polyribonucleotide.

-   [136] The composition of any one of paragraphs [93] to [135],    wherein the circular polyribonucleotide comprises a plurality of    expression sequences having either a same nucleotide sequence or    different nucleotide sequences.

-   [137] The composition of any one of paragraphs [93] to [136],    wherein the circular polyribonucleotide is substantially resistant    to degradation, e.g., exonuclease.

-   [138] The circular polyribonucleotide of any one of paragraphs [94]    to [137].

-   [139] A method of producing the composition of any one of paragraphs    [93] to [137], comprising combining the circular polyribonucleotide    of any one of paragraphs [93] to [137] and the pharmaceutically    acceptable carrier or excipient of any one of paragraphs [93] to    [137].

-   [140] A method of treatment, comprising administering the    composition of any one of paragraphs [93] to [137].

-   [141] A method for protein expression, comprising translating at    least a region of the circular polyribonucleotide of any one of    paragraphs [93] to [137].

-   [142] The method of paragraph [141], wherein the translation of the    at least a region of the circular polyribonucleotide takes place in    vitro.

-   [143] The method of paragraph [141], wherein the translation of the    at least a region of the circular polyribonucleotide takes place in    vivo.

-   [144] A polynucleotide encoding the circular polyribonucleotide of    any one of paragraphs [65] to [137].

-   [145] A method of producing the circular polyribonucleotide of any    one of paragraphs [65] to [137].

-   [146] The method of paragraph [145], comprising splint    ligation-mediated circularization of a linear polyribonucleotide.

-   [147] The method of paragraph [146], wherein the splint    ligation-mediated circularization has an efficiency of at least 2%,    at least 5%, at least 10%, at least 15%, at least 20%, at least 25%,    at least 30%, at least 32%, at least 34%, at least 36%, at least    38%, at least 40%, at least 42%, at least 44%, at least 46%, at    least 48%, or at least 50%.

-   [148] The method of paragraph [146], wherein the splint    ligation-mediated circularization has an efficiency of about 40% to    about 50% or more than 50%.

What is claimed is:
 1. A method of expressing a polypeptide in asubject, comprising: administering to the subject a covalently closedpolyribonucleotide that: (a) comprises an internal ribosome entry site(IRES) element; (b) comprises an expression sequence that encodes thepolypeptide and that lacks one or both of a 5′ cap and a poly-Asequence; and (c) comprises a termination element; and translating thepolypeptide from the expression sequence of the covalently closedpolyribonucleotide in vivo over to of at least 7 days.
 2. The method ofclaim 1, wherein the polypeptide is a therapeutic polypeptide.
 3. Themethod of claim 1, wherein the covalently closed polyribonucleotidefurther comprises at least one element selected from: (a) anencryptogen; (b) a regulatory element; and (c) a quasi-double-strandedsecondary structure.
 4. The method of claim 1, wherein the internalribosome entry site element comprises a sequence derived frompicornavirus complementary DNA, encephalomyocarditis virus (EMCV)complementary DNA, poliovirus complementary DNA, or an Antennapedia genefrom Drosophila melanogaster.
 5. The method of claim 1, wherein thetermination element comprises a stop codon.
 6. The method of claim 1,wherein the covalently closed polyribonucleotide comprises two or moreof the expression sequence encoding the polypeptide.
 7. The method ofclaim 1, wherein the covalently closed polyribonucleotide furthercomprises a second expression sequence encoding a second polypeptide. 8.The method of claim 1, wherein the polypeptide comprises at least 150amino acids.
 9. The method of claim 1, wherein the polypeptide is anantigen.
 10. The method of claim 9, wherein the antigen is a viralantigen, a bacterial antigen, or a tumor antigen.
 11. The method ofclaim 1, wherein the polypeptide is at least a functional portion of aviral envelope protein.
 12. The method of claim 1, wherein thepolypeptide is selected from the group consisting of a hormone, acytokine, a ligand, a receptor, an antibody, and an enzyme.
 13. Themethod of claim 1, wherein the polypeptide is a secreted protein. 14.The method of claim 1, wherein the polypeptide is an epigeneticmodifying agent.
 15. The method of claim 1, wherein the polypeptide isan epigenetic enzyme.
 16. The method of claim 1, wherein the polypeptideis a nuclease.
 17. The method of claim 1, wherein the polypeptide is acomponent of a CRISPR system.
 18. The method of claim 1, wherein thepolypeptide is a nuclease and the covalently closed polyribonucleotidefurther comprises a guide RNA sequence.
 19. The method of claim 1,wherein the polypeptide is selected from the group consisting of a poreforming peptide, a cytotoxic peptide, and an anti-microbial peptide. 20.The method of claim 1, wherein the covalently closed polyribonucleotidefurther comprises an encryptogen.
 21. The method of claim 20, whereinthe encryptogen comprises a splicing element, a modified ribonucleotide,or a protein binding site.
 22. The method of claim 20, wherein theencryptogen comprises an immunoprotein binding site.
 23. The method ofclaim 1, wherein the covalently closed polyribonucleotide is formulated,for the administration, with: a) a pharmaceutically acceptableexcipient; b) a polymeric carrier; c) an exosome; d) a lipid carrier; ore) a lipid nanoparticle.
 24. The method of claim 1, wherein thecovalently closed polyribonucleotide further comprises a miRNA targetsequence.
 25. The method of claim 1, further comprising detectingexpression of the expression sequence in a cell or a tissue of thesubject before and/or after the administration.
 26. The method of claim1, further comprising detecting the polypeptide in the subject over atime period of at least 7 days.
 27. The method of claim 1, furthercomprising detecting the polypeptide in the subject over a time periodof at least 21 days.
 28. The method of claim 1, wherein the subject is ahuman.
 29. The method of claim 1, wherein the subject is a non-humanmammal.
 30. A method of delivering a therapeutic polypeptide to a human,comprising: administering to the human a covalently closedpolyribonucleotide that: (a) comprises an internal ribosome entry site(IRES) element; (b) comprises an expression sequence that encodes thetherapeutic polypeptide and that lacks one or both of a 5′ cap and apoly-A sequence; and (c) comprises a termination element; andtranslating the therapeutic polypeptide from the expression sequence ofthe covalently closed polyribonucleotide in vivo over a period of atleast 7 days, wherein the therapeutic polypeptide is selected from thegroup consisting of an antigen, at least a functional portion of a viralenvelop protein, a hormone, a cytokine, a ligand, a receptor, anantibody, an enzyme, a secreted protein, an epigenetic modifying agent,a nuclease, a component of a CRISPR system, a pore forming peptide, acytotoxic peptide, and an anti-microbial peptide.